A detailed review of bovine brucellosis

Abstract

Bovine brucellosis, a prevalent zoonotic disease, is caused by the bacterium Brucella abortus, which can infect both humans and animals. This review article aims to provide a full explanation of the causes, historical background, occurrence, development, clinical manifestations, diagnosis, spread, variables that increase the risk, importance to public health, economic impact, treatment, and methods of controlling bovine brucellosis. Bovine tuberculosis primarily affects cattle although it can also affect other animals. Bovine brucellosis is a global illness that occurs in all regions of the world, with the exception of Antarctica. Cattle afflicted with bovine brucellosis may suffer from persistent and incapacitating illness. The main techniques used to identify Brucella infection are serological assays, such as the Rose Bengal plate test, milk ring test, serum agglutination test, and enzyme-linked immunosorbent assay. The prevalence of Brucellosis exhibits significant variation as a result of disparities in the precision and efficacy of serological tests. Brucella abortus can be found in various physiological fluids and tissues, such as milk, urine, feces, vaginal secretions, semen, bones, joints, male reproductive organs, placenta, and the fetuses of pregnant women. This condition commonly decreases cow productivity and can negatively impact the financial performance of livestock businesses, particularly dairy sectors. The most effective initial treatment for brucellosis is isoniazid, doxycycline, and streptomycin. The vaccine strains most frequently employed to protect cattle against Brucella infection and related abortions are strains 19 and RB51. Research has shown that bacteriophage lysates are effective for treating bovine brucellosis. Adopting a One Health strategy can help effectively treat this illness by considering the health of both animals and humans.

Introduction

Bovine brucellosis remains a significant public health problem in several countries, with multiple economic and zoonotic implications. This zoonosis is responsible for substantial reproductive disorders and loss of cattle production. Multiple countries regulate surveillance and control under specific conditions to obtain and maintain their official free status, facilitating access to export markets. According to Schelling et al. (2003), the World Health Organization (WHO), the Food and Agriculture Organization (FAO), and the Office International des Epizootics (OIE) regard Brucellosis as one of the most widespread zoonosis. Almost every domestic animal is susceptible to Brucella infection, except cats, which have developed natural immunity. In addition, it can potentially infect humans, other ruminants, and aquatic animals. Epizootic abortion, Bang’s disease, undulant fever, Malta fever, and contagious abortion are some of the well-known names for brucellosis (Roberts and Kemp, 2001).

According to Kurdoglu et al. (2015) and Lokamar et al. (2020), brucellosis is a serious zoonotic disease that has the potential to result in considerable fertility losses in sexually developed animals. The disease primarily results in abortion during the final trimester of pregnancy, stillbirth, placental and testicle inflammation, infertility, and the presence of the organism in uterine discharges and milk (Reddy and Sivajothi, 2024). According to figures, the losses resulting from culling as a result of this disease can account for 30%–50% of the difference between the market value of dairy or beef cattle and their value at the time of slaughter (Refai, 2002; Bardhan et al., 2020). The intake of raw or unpasteurized milk and animal products and contact with infected placenta or carcasses are major factors contributing to zoonotic bovine Brucellosis infections in humans (Pal et al., 2017; Almashhadany et al., 2022). Identifying the precise prevalence of bovine brucellosis caused by Brucella abortus in humans is challenging because of technological obstacles in isolating the pathogen. The presence of humans and animals in the same environment, especially in rural regions, has led to a rise in the incidence of bovine brucellosis in humans in developing nations (Godfroid et al., 2013).

Brucellosis is classified as an occupational disease for individuals working in agriculture, livestock, slaughterhouses, veterinary medicine, meat inspection, and laboratory personnel (Pereira et al., 2020; Sadighi et al., 2020). Individuals who consume dairy products and come into contact with animals and animal corpses in areas where there is a high prevalence of infection are at a significantly higher risk of developing brucellosis (Dadar et al., 2019; Milton et al., 2020). Brucella spp. take advantage of the host’s immunological defenses to form long-lasting infections, resulting in a range of clinical symptoms that vary from fever, tiredness, and joint pain to more serious sequelae like endocarditis and neurological problems (Pellegrini et al., 2022). The diagnosis of brucellosis requires clinical assessment and laboratory investigations, such as blood culture, serological testing, and molecular techniques. However, diagnosing the condition is difficult because the symptoms are not specific, and it is challenging to obtain appropriate samples for testing (Padilla Poester et al., 2010).

Treatment usually consists of a combination of several medications. However, the development of antibiotic-resistant Brucella strains poses considerable challenges (Dafale et al., 2020). Administering vaccines to animals that have the potential to carry germs, particularly in cattle herds, shows promise in limiting the transmission of brucellosis (Khurana et al., 2021). Despite extensive efforts to control and alleviate the spread of brucellosis, it remains a substantial obstacle to public health (Moriyón et al., 2023). This study offers a comprehensive examination of brucellosis, with a specific focus on its epidemiology, pathogenesis, diagnostics, and therapeutic approaches. Additionally, it provides up-to-date information on the most recent studies on Brucella species, including their range of hosts and the many ways in which they are transmitted.

The purpose of this review was to provide a comprehensive overview of bovine brucellosis, including its background, symptoms, diagnosis, risk factors, economic impact, treatment choices, management, and public health importance.

Etiology

Brucella abortus is the primary cause of brucellosis in cattle, although other Brucella species, such as B. melitensis and B. suis, can infect calves (Olsen and Tatum, 2010). Brucella spp. typically exhibit a preference for certain primary hosts, but rarely restrict their infection to just one type of host. The scope of this discussion will be restricted to B. abortus. B. abortus is a small, gram-negative bacterium that requires certain conditions and thrives in an environment with high levels of carbon dioxide and oxygen (Jamil et al., 2017; Barbier et al., 2018). The bacterium has numerous intricate prerequisites for growth in a laboratory setting, and differentiating between Brucella species and identifying biotypes is challenging. The techniques used for speciation and biotyping encompass CO₂ demands, H2S generation, cultivation in diverse colors, bacteriophage lysis, substrate oxidation, and agglutination assays (Araj, 2010). Various biotypes of B. abortus have been recognized, but in the United States, biotype 1 is the predominant type and biotypes 2 and 4 have lesser significance (Bricker and Ewalt, 2005; Martínez et al., 2014). Although organisms have intricate developmental needs under laboratory conditions, they may survive for extended periods in specific animal products and the environment under favorable conditions (Kaden et al., 2018).

Typically, the creature thrives in environments with high moisture levels and cool temperatures, but it does not thrive well in conditions of sunlight, dryness, and heat. For instance, B. abortus can endure in manure at a temperature of 12°C for 250 days, but it is rapidly destroyed in hot manure (Sobsey et al., 2006; Polley et al., 2022). Furthermore, it is worth noting that contaminated placenta and fetal tissues, refrigerated infected milk and other dairy products, as well as chilly water, have the potential to sustain infectivity for an extended period (Corbel, 2006). The process of pasteurization effectively eliminates B. abortus bacteria present in milk (Olsen et al., 2017). The most common route of infection is swallowing the organism; however, it can also spread through the vagina, uterus, and birth canal on rare occasions. Infected placental fluids, tissues, fetuses, or milk can contaminate feedstuffs, pasture, or fomites, making sensitive cattle more likely to ingest the organism (FEVER & FEVERS, n. d.). Epidemic and persistent endemic diseases in dairy cattle are more likely in large herds managed intensively.

The organism becomes a facultative intracellular host once infection occurs, allowing it to survive in the host’s phagocytic cells (Freddi et al., 2023). The cell wall’s polysaccharides and lipopolysaccharide (LPS) proteins are surface antigens that cause the host to produce agglutinating antibodies. Surveillance programs for brucellosis rely on serologic testing that can detect these antibodies. Given B. abortus’s facultative intracellular classification, it is not surprising that cell-mediated immunity is significant yet poorly understood. Bacillus abortus infects cattle via cuts, holes in the skin, or mucous membranes of the mouth, nose, and conjunctiva. The pharynx is believed to be the principal entrance site because swallowing is the most common route of infection spread. Infected milk can infect calves, and the disease can pass from cows to bulls through the vaginal canal (Ficht, 2003).

History

Around 450 B.C., Hippocrates was the first to record a case of recurrent fever and mortality that lasted four months. The arrival of undulant fever in America was not until 1905 when the S.S. Adriatic brought 65 Maltese goats (Tripp and Sawchuk, 2011). In 1914, Traum in the United States isolated B. suis from abortion pigs in Indiana. Isolation of B. ovis from sheep infected with ram epididymitis in Australia and New Zealand occurred in 1953 (Cerri et al., 2002). Dogs, caribou, and reindeer were the first B. canis discovered in 1966 (Davis, 1990). In 1530, the Knights of the Order of St. John were most graciously granted the island of Malta. The records of infectious fevers date back to that era and continued until the nineteenth century. The numerous reports of undulant fevers that appeared in the Mediterranean in the 17th and 18th centuries were based on a variety of regional names, including Mediterranean fever, Rock fever of Gibraltar, Cyprus fever, and Danube fever (Fever et al., 2012). In 1810, British naval surgeon Sir William Burnett made history by cataloging the various fevers that afflicted sailors in the Mediterranean. Local medical experts’ proficiency in using clinical thermometers to trace the progression of undulant fever, along with the large number of British troops that came to Malta for recuperation during the Crimean War (1853–1856), likely played a role in the island’s rise to prominence as a center for undulant fever research (Shepherd, 1991).

Following the contraction of Malta fever, British army surgeon J. A. Marston meticulously recorded a comprehensive account of the patient’s condition, marking the first instance of such documentation. He had muscle and joint problems, gastrointestinal disorders, and intermittent fever that persisted for 30–90 days. Sir David Bruce, a British Army medic, successfully identified the specific microorganism responsible for Malta fever on July 9, 1887 (Cook, 2007). He officially termed this microbe Micrococcus melitensis. A British soldier who succumbed to the illness had it extracted from his spleen. In addition, he observed that the organism flourished at higher temperatures, which led him to hypothesize that this could explain the increase in cases during the summer. Upon successfully identifying the bacterium in goat blood, urine, and milk, the researcher concluded that goats were the primary source of infection. The discovery enhanced our understanding of the disease’s epidemiology. For instance, officers had a tripled risk of disease compared to private troops because they consumed milk, and hospitals that distributed milk had a large number of cases (Bruce, 1889).

In 1897, during an extensive investigation spanning over a century, the Danish physician and veterinarian Bernhard Bang discovered the presence of Bacterium abortus while studying the transmission of viral abortion in cattle in Denmark. Furthermore, he discovered that the organism had an impact on sheep, goats, horses, and sheep. Consequently, the disease was named “Bang’s disease” Williams and McKusick, 1954). Alice Evans, an American bacteriologist, discovered the connection between animals and people during the 1920s. Both Bang’s Bacterium abortus and Bruce’s Micrococcus melitensis exhibited notable similarities in their physical structures and disease-causing abilities. The contemporary nomenclature of these organisms continues to employ the appellation of Sir David Bruce (Moreno, 2021).

Epidemiology

Brucellosis is prevalent throughout the world, except in Antarctica. Approximately 60 million cattle are believed to be susceptible to this disease. Brucellosis is an infectious disease caused by many Brucella bacteria that can infect humans and animals, including cattle, dogs, sheep, and goats (Hall, 2020). Recent research suggests that the global occurrence of this phenomenon is more widespread than previously thought, with an approximate annual incidence of 1.6–2.1 million new instances in humans (Laine et al., 2023). Regions characterized by scarce resources, such as the Mediterranean, Middle East, Central Asia, and specific areas of Africa, exhibit greater occurrence rates (Qureshi et al., 2023). Brucella canis has recently become a significant source of canine brucellosis in Europe, posing a potential hazard to public health due to its ability to be transmitted between animals and humans. The limited scope of surveillance and awareness of B. canis among veterinarians and dog owners hinders the effective management of the disease (Djokic et al., 2023).

Despite its previous prevalence, bovine brucellosis has been effectively or nearly eradicated in cattle populations in numerous countries through management programs. Based on recent statistics, the following countries are free from bovine brucellosis. Australia, New Zealand, Canada, Japan, and 16 member states of the European Union (EU) have been formally recognized as being free from brucellosis, according to Cárdenas et al. (2019). Efforts are being made to eliminate bovine brucellosis in the United States, as well as in many European countries (Borham et al., 2022). Bovine brucellosis, while capable of infecting many livestock species such as goats, sheep, and camels, as well as wild animals including civets, possums, and deer, remains a significant infectious disease that primarily impacts cattle populations in some regions (Bengis et al., 2004; Kosoy and Goodrich, 2019). In countries with limited development and insufficient control measures, the disease remains a threat to public health and presents significant economic difficulties for the livestock sector (Terefe et al., 2017).

The emergence of Brucella canis as a cause of canine brucellosis in Europe is concerning from a public health perspective, as it has the potential to be transmitted to people (Buhmann et al., 2019). The inadequate extent of surveillance and understanding of B. canis among veterinarians and dog owners hinders the efficient control of the disease. The current diagnostic tools for detecting B. canis infection do not have sufficient sensitivity and specificity, emphasizing the need for enhanced diagnostic techniques (De Oliveira et al., 2011). The lack of globally applicable reagents and standards for serological testing hinders the accurate diagnosis of this infection. To address these issues, this study emphasized the importance of developing informative materials, specialized guidance, and enhanced diagnostic techniques to manage the spread of B. canis and increase awareness among the general public and experts. Bovine brucellosis appears to differ considerably between regions within a country. Reports from South America suggest that locations located near densely populated towns with a high concentration of intensive milk production experience the greatest rates of bovine brucellosis (Thimm, 2013; Moriyón et al., 2023). The occurrence of bovine brucellosis in the sub-Saharan African region varies from 0% to 55.8%. Subsequently, the regions of North America and Western Europe exhibit a prevalence rate of 31.1%, whereas India demonstrates a prevalence rate of 15.1% (Qureshi et al., 2023).

In countries with sophisticated testing and control systems, as well as a comprehensive set of monitoring and control procedures, bovine brucellosis is likely to be considered an infectious disease with low occurrence and a seemingly low rate of transmission (Robinson and Production, 2003). Cattle are the main reservoirs for bovine brucellosis (Zheludkov and Tsirelson, 2010). Bovine brucellosis infections mainly affect individuals who consume unpasteurized milk and dairy products. The bacteria responsible for Brucellosis can affect various parts of the body, such as the reproductive system, liver, heart, and central nervous system (Jawad, 2024). Chronic brucellosis can lead to difficulties in either a single organ or throughout the body. The potential consequences may involve inflammation in the inner lining of the heart chambers, a condition known as endocarditis (Kamde and Anjankar, 2022).

Pathogenesis

A number of factors influence the disease prognosis; these factors include LPS, type IV secretion system (T4SS), and BvrR/BvrS. When bacteria replicate, these parts help them come into contact with host cells, form Brucella-containing vacuoles (Bcvs), and interact with the endoplasmic reticulum (ER). Brucellosis has a complex pathophysiology that includes immune evasion, persistent infections, and bacterial penetration into host cells. Brucella uses tactics to evade host immune defenses, allowing the infection to persist for an extended period. The bacterium can infiltrate and remain within host cells, including macrophages. Common symptoms in humans include tiredness, high body temperature, and overall unease, as well as more serious problems, such as joint inflammation, bone infection, heart valve inflammation, and the brain and its surrounding membranes (Coelho et al., 2019; Wardhan, 2020).

Brucella can thrive and multiply within host cells, thereby evading the immune system. This is an extremely proficient intracellular pathogen. These compounds impede the phagocytosis process, reduce the ability to kill bacteria, mitigate the intensity of endotoxic reactions, and obstruct the presentation of antigens. The pathogenesis of Brucella involves its ability to endure and reproduce inside cells that can engulf foreign particles and cells that cannot, as well as its ability to alter the functions of host cells, hinder the functioning of cells that can engulf foreign particles, inhibit the engulfment of foreign particles by cells, and impede the programmed cell death of host cells. One example of how the immunological response of the host is affected by the degradation of the tlR signaling adapter MAl is through the targeting of critical signaling pathways in innate immunity (Byndloss et al., 2016; Głowacka et al., 2018; Elrashedy et al., 2022). Brucella’s capacity to endure various environmental conditions, including water, soil, dairy products, and meat, is an additional feature that enhances the disease’s ability to cause illness and spread.

Clinical symptoms

Cattle affected with bovine brucellosis exhibit a number of obvious signs, such as abortion, giving birth to weak calves, and vaginal discharge. It is common for cows infected with the disease to experience abortions between the fifth and seventh month of their pregnancies, although this is not the case for all cows infected. It is possible for infected cows to contain and excrete infectious germs in their milk and uterine secretions throughout their life, even if their calves appear to be in good health. Other indications include prolonged presence of placentas, infections in the uterus, low fertility rates, hygromas, decreased milk output in cows, and orchitis in bulls. All these symptoms are associated with preterm labor. It is possible for horses to acquire infection caused by B. abortus by cattle. This infection can lead to the development of fistulous withers and pollutant evil, both of which are exceptionally challenging to treat.

Fitch et al. examined 41 uteri during pregnancy and discovered that B. abortus was absent from the uteri prior to the 4.5-month mark. However, they were able to remove the organism from 13 of the 29 uteruses that had gestations lasting longer than 4.5 months. Infected cows gain immunity, and Bang discovered that infected pregnant females usually have a single abortion. In most cases, the organs are unaffected by brucellosis, but a small amount of inflammation in the mammary gland’s intercellular tissue is occasionally noticeable. The process of removing bacteria from milk is associated with this inflammation. In chronically infected cows, macrophages in the mammary gland may provide an ideal environment for the survival of B. abortus. Prepubescent heifers are susceptible to the virus, but infected people have not shown signs of illness.

A study conducted by (Edgington and Donham, 1939) revealed that 15 heifers infected with B. abortus before breeding did not experience abortions during their subsequent pregnancies. Nevertheless, there is evidence to suggest that heifers infected before they reach maturity may have abortions and contribute to the infection’s spread within the herd (Luna et al., 2022). It is widely recognized that bulls are adversely affected by B. abortus infection. Schroeder and Cotton (1917) identified the causative agent by investigating the body of a bull with an abscess in the epididymis and isolated the same bacteria from bulls with seminal vesiculitis and/or orchitis. Orchitis, epididymitis, ampullitis, and seminal vesiculitis may be a consequence of reproductive system infection. Orchitis is uncommon, and when it does manifest, it frequently impacts only one testis; however, both testicles may be vulnerable. The complete mortality of testicular tissue is a consequence of the merging of dispersed areas of cell death. Furthermore, testicular atrophy is possible. It is hypothesized that the syndrome involving the seminal vesicles and ampoules is likely to occur more frequently than that involving the testicles and epididymides. The incubation time of brucellosis in bulls has not been precisely determined. After vaccination, we observed orchitis caused by strain 19 of B. abortus 10 days later. Danks discovered orchitis caused by the same strain seven months later.

Lopes et al. (2010) observed no noticeable clinical changes in three young male cattle when they were repeatedly exposed to B. abortus at three different times over a period of 6 months. The time taken for orchitis to develop after infection in a single bull was at least 122 days. Hygromas and arthritis are occasionally detected (Musa et al.,1990). Brucellosis can cause pneumonia in the fetus, which is characterized by the consolidation of the lungs and the appearance of a yellowish mottling appearing in some fetuses (Laine et al., 2023). However, it is important to recognize that not all aborted fetuses with brucellosis develop pneumonia, and the appearance of lung lesions alone is not enough evidence to link B. abortus to the abortion (Leekha et al., 2011). Aborted fetuses often have noticeable extra abnormalities. The findings of Li et al. (2014) indicated that intentionally infecting heifers with B. abortus resulted in the birth of weak newborn calves or the termination of pregnancy with moderate inflammation of the abdominal lining and a small accumulation of fibrinous fluid on the abdominal organs. Furthermore, the exudate was identified within the pericardium.

Clinical diagnosis

The clinical manifestations of this illness can range from moderate symptoms such as fever, fatigue, and joint pain to more severe problems such as endocarditis and brain abnormalities (Lindén et al., 2014). Brucellosis diagnosis requires clinical assessment and laboratory analyses, including blood cultures, serological tests, and molecular approaches. Nevertheless, diagnosis is challenging due to the lack of identifiable symptoms and difficulty in obtaining suitable specimens for testing (Lokamar et al., 2020; Luna et al., 2022).

Postmortem diagnosis

A common symptom of developing lesions is cotyledon necrosis. Fetuses that do not survive the abortion process commonly present with pneumonia symptoms and edema. Infected bovine testicles show signs of necrosis (Ma et al., 2022). A buildup of fluid, known medically as edema, can cause lymph nodes to swell and grow, or they can remain of normal size. A hallmark of septicemia is hyperplasia, an abnormally high rate of cell reproduction; this swelling, however, is not a hallmark of septicemia (Manish et al., 2013). For specific areas of cells to die, necrosis is necessary. Liver disease in cattle. Sometimes, parenchymatous organs, including the kidneys, liver, and heart, can show signs of degeneration (Mariana et al., 2010). The occurrence of additional observable defects in aborted fetuses is extremely frequent. It is found that cows infected with B. exhibited persistent reproductive problems. The aborted fetus had characteristics of vulnerable newborn calves or deceased fetuses, displaying moderate peritonitis and a layer of fibrinous exudate on the abdominal organs and pericardium (Megid et al., 2010; Martínez et al.,2014).

Rose Bengal Test

The primary purpose of the Rose Bengal test is to assess the potential risk of infection among different demographic groups, particularly in epidemiology fields. In the veterinary domain, this technique is employed as a screening method in regions with a low incidence of illness and as a diagnostic tool in cattle and pig populations with a relatively high prevalence of brucellosis. Validating a preliminary diagnosis, which is made based on the patient’s medical history and clinical observations, heavily relies on conducting antibrucella antibody tests. The Rose Bengal test (sometimes referred to as the buffered test) is a rapid slide agglutination procedure used to specifically detect Brucella antibodies in human and animal sera. The bacterial suspension is reactive to immunoglobulin M and G antibodies. In comparison with the usual tube agglutination test, the former is capable of detecting infections at an earlier stage (sub-clinical infections) and for a longer duration of the disease (chronic stage). The assay evaluates the pH 3.6 buffered suspension of the B. abortus strain, which is colored with Rose Bengal, against sera of unknown origin. In the analyzed samples, the presence or absence of visible clumping indicates the presence or absence of antibodies (Miller and Kaneene, 2006; Mesner et al., 2007; Megid et al., 2010).

Milk ring test

The primary site of Brucella antibody absorption in bovine milk is fat globules. Upon exposure to milk containing specific antibodies and an antigen stained with hematoxylin, the antigen-antibody response is observed as a color change in the milk. The formation of a ring cream on the surface of the milk is a consequence of the color change that occurs during the incubation period. The milk ring test is the most efficient approach for monitoring brucellosis-free herds and identifying infected dairy cows. We added 30 L (0.03 milliliters) of B. abortus Bang Ring Antigen, a hematoxylin-stained, to the test. The milk column was maintained at a height of 25 mm. In addition to the positive and negative control samples, the milk (antigen) mixes were incubated at 37°C for 1 h. The upward movement of the fat globules resulted in the aggregation of Brucella cells, forming a dense layer of cream on the sample surface. The appearance of a vivid blue circle atop a white milk column indicates a highly favorable reaction. The milk under the cream layer became acidic when its color surpassed that of the cream layer, although the cream layer appeared normal. The observed color intensity of the cream layer facilitated the categorization of the samples as negative, 1+, 2+, 3+, or 4+. The milk ring test is a cost-effective method for monitoring brucellosis in dairy herds. Milk and whey samples have been widely utilized for the antibody screening in herds or individual animals, making them valuable and easily obtainable (Mittal and Tizard, 1983; Mohamand et al., 2014; Milton et al., 2020; Moreno, 2021).

Standard Tube Agglutination Test

The standard tube agglutination test (STT) was used to detect antibodies against Brucella abortus, the causative agent of bovine brucellosis. Use a plain tube without any additional substances like a serum separator or a red cap, to separate serum from clotted blood. If feasible, transfer the clear serum from the clot to a fresh tube. There were 10 test containers in a rack, each measuring 13 x 100 mm. The initial test tube was subsequently filled with 0.9 ml of saline solution, while the remaining test tube was filled with 0.5 ml. Then, the initial test equipment was introduced with 0.1 ml of the tested serum. After mixing, 0.5 ml of the weakened serum was transferred to a second test tube. Afterward, 0.5 ml of the diluted serum was transferred from the second to the third test tube. This process was repeated until tube 10 was merged. The initial procedure involved discarding 0.5 ml of the diluted serum. The dilutions in the 10 test tubes were varied from 1:10 in tube number 1 to 1:5120 in tube number 10. To serve as antigen control, an extra tube was included in the series, which contained 0.5 m1 of saline. A 0.5 m1 solution of B. abortus antigen was diluted in saline at a ratio of 1:50. The diluted solution was then added to each tube, resulting in final dilutions ranging from 1:20 to 1:10240. The rack was then placed in a container of water that was maintained at 37°C for 48 hours while being vigorously stirred. Simultaneously, the same technique was applied to both the positive and negative controls (Musa et al., 1990; Megid, 2010; Moriyón et al., 2023; Munir et al., 2023)

Indirect enzyme-linked immunosorbent assay

The immunological test I-ELISA was used to identify Brucella antibodies that specifically targeted the LPS antigen of B. abortus, B. melitensis, and B. suis (Njenga et al., 2020). All serum samples, controls, and reagents were uniformly adjusted to ambient temperature (18°C–25°C). The sera and control samples were diluted at a ratio of 1:20 and incubated at room temperature for 45 minutes with plates coated with B. abortus LPS. After washing, each well of the microplate was treated with a 100-μL solution of a multispecies horseradish peroxidase (HRP) conjugate and incubated at room temperature for 30 minutes. After eliminating the excess conjugate by washing, a solution of Tetra methyl Benzidine (TMB) substrate was added and allowed to sit in darkness for 15 minutes. The color that is produced is determined by the concentration of the specific antibody in the analyzed material. The plates were ultimately analyzed using an ELISA microplate reader operating at a wavelength of 450 nm (Ollé-Goig and Canela-Soler, 1987; Olsen and Tatum, 2010; Padilla Poester et al., 2010; Olsen et al., 2017; Olsen et al., 2018; Njenga et al., 2020; Otaraeva et al., 2022).

Brucella coombs Gel Test

Brucella antibodies were introduced into the diluted serum samples on dilution plates. The samples were transferred into the 12 × 8 gel microtubes using a pipette together with an antihuman IgG gel matrix. The outcomes were assessed according to agglutination following centrifugation. The object was rotated at a speed of 3000 revolutions per minute for a duration of 20 minutes. If the pink-hued Brucella antibody settled at the bottom of the microtubes, the samples were designated as negative; conversely, if the pink-hued antibody remained suspended above the gel, the samples were designated as positive (Palmer et al., 2012; Pal et al., 2017).

Complement fixation test

The recognition of the complement fixation test (CFT) as a reliable method for diagnosing bovine brucellosis does not imply that laboratories universally adhere to identical protocols. This study conducted experiments using microtitration plates and an 8-channel microdilutor, which was operated based on the principles of peristaltic pump theory. The serum was diluted using this equipment, and the reagent was also dispensed. The complement was treated with Richardson’s preservative and subsequently analyzed using spectrophotometry to determine the 50% hemolytic dose (C’H50). The test includes three C’H50 assays. The experiment utilized a high-quality supplement that enabled dilution in the range of 1/60–1172. Rabbits were used to generate hemolysin with a minimum titer of 1/2000. The test consists of five minimal hemolytic doses (MHOs) of hemolysin. The serum dilutions increased twofold, with the 1/2 dilution serving as an anticomplementary control. The actual examination was conducted using serum dilutions ranging from 1/4 to 1/128 (Paweska et al., 2002; Pappas et al., 2006; Pal et al., 2020)

Molecular Detection

Our technique effectively detects brucellosis in humans and animals, even when its symptoms are unknown or missing. However, a positive result can indicate the presence of genetic material from dormant or treated bacteria, which does not necessarily imply an ongoing infection. Although serological tests and sensitive nucleic acid amplification are sufficient for identifying brucellosis, culture remains the most precise approach due to its extensive clinical and epidemiological significance. Peripheral blood is the most advantageous sample for molecular research on human brucellosis. Even if cultures yield negative results, obtaining additional samples from other systems can help diagnose localized brucellosis. Genetic materials extracted from tissues preserved in formalin and embedded in paraffin can be analyzed using established procedures.

Different gene targets have been used to identify Brucella infections. A diagnostic target in the 16S rRNA gene could be identified. Still, there have been past incidents of possible cross-reactivity that may have provided erroneous positive results. Consequently, one could consider the iS711 insertion sequence as a potential goal. However, the use of this tool has been questioned given alterations in the sequence and its absence in some strains of Brucella, which makes it untrustworthy in some contexts. Moreover, because of its capacity to generate an immunogenic membrane protein, BCSP31 is often used for diagnostic purposes. The species-specific quantitative polymerase chain reaction (qPCR) assays, together with traditional Brucella ladder PCR assays, are essential for the precise identification and categorization of Brucella species. The MlvA-16 panel is a reliable diagnostic tool for detecting human brucellosis. This panel specifically targets 16 unique genetic regions, making it very specific to the disease. PCR-based techniques offer accurate and highly sensitive identification of Brucella. These procedures are essential for establishing the presence of the disease and identifying the particular species responsible for it. Several amplification techniques have been used, such as real-time polymerase chain reaction (PCR), multiplex PCR, nested PCR, PCR-enzyme immunoassay (PCR-EIA), and loop-mediated isothermal amplification method (LAMP). The nested polymerase chain reaction (PCR) enhances the accuracy and sensitivity of detection by utilizing two sets of primers in two consecutive amplification cycles. To enhance detection sensitivity, the PCR-EIA method was combined with an enzyme immunoassay performed in a microplate configuration. The LAMP technique offers various benefits in situations where resources are scarce, such as its simplicity, quick reaction time, and cost-effectiveness.

Transmission

The vast majority of information regarding the transmission of zoonotic Brucellosis comes from research on brucellosis infection in bovines. It is possible for these germs to be detected in respiratory secretions, milk, vaginal secretions, sperm, urine, feces, and exudates from lesions (such as lymph node drainage and some skin lesions), depending on the location of the lesions. All links in the meat and dairy supply chains must implement stringent safety procedures to prevent the spread of the disease, since the majority of cases involve the consumption of raw or undercooked meat and unpasteurized dairy products. The fact that Brucella species can survive in a variety of food processing environments does not mean that you can avoid not fully cooking anything before eating it (Pellegrini et al., 2022).

Brucellosis is mainly transmitted by direct contact with infected animals or their bodily fluids, including vaginal discharges, aborted tissue, and sperm (Peña Joya et al., 2011). This is the primary transmission mode. Individuals who have regular and intimate interactions with livestock, such as farmers, veterinarians, and livestock managers, are at a higher risk of developing the disease owing to their frequent exposure to animals (Pereira et al., 2020). This is a consequence of the increased probability of interaction with animals.

Consuming raw or unpasteurized dairy products derived from infected animals, such as milk and cheese, can lead to the transmission of the brucellosis virus. Ingesting these tainted foods can lead to illness in humans, underscoring the importance of implementing food safety regulations to mitigate the transmission of the illness (Plackett and Alton, 1975; Plant et al., 1976).

The spread of Brucella bacteria through the air can be an issue in certain work situations, such as slaughterhouses and meat processing facilities. People who work in such environments have a risk of being exposed to airborne germs (Poester et al., 2013), which could lead to the development of an infection. This demonstrates the importance of designing efficient protocols for workplace safety and using protective gear that is suitable for every situation. Occupational exposure to diseased animals or their products, such as in butchering, laboratory work, and hunting, significantly increases the risk of developing human brucellosis. It is important to undertake occupational health precautions to mitigate the impact of this risk (Polley et al., 2022).

Human infections obtained in laboratories related to brucellosis are commonly encountered (Qureshi et al., 2023). For example, a study discovered that 12 out of 48 healthcare workers in a hospital in Ankara were diagnosed with Brucella species, leading to an infection risk of 8% per worker annually (Qureshi et al., 2023). Although the transmission of brucellosis between individuals is rare, it is crucial to be knowledgeable about alternative routes of transmission. Blood transfusions and bone marrow transplants are the recommended procedures, emphasizing the importance of antibody detection technologies, especially in countries with a high prevalence of the disease (Reddy and Sivajothi, 2024). Furthermore, brucellosis can be transmitted via the inhalation of tiny airborne particles, direct contact with infected skin, and colonization of the udder, which occurs when contaminated milking equipment is utilized (Revich et al., 1961; Refai, 2002). Brucellosis is a probable type B bioweapon (Roberts and Kemp, 2001; Robinson and Production, 2003).

Furthermore, improper treatment of milk, dairy products, and meat has played a role in the transmission of human brucellosis, highlighting the ability of the illness to be transmitted from animals to humans (Sadighi et al., 2020). Ultimately, occupational exposure is a significant barrier to the spread of brucellosis. Professionals in specific industries must be vigilant and implement essential procedures to reduce infection risk. Furthermore, public health activities must prioritize the mitigation of these possible transmission sources (Sadusk et al., 1957). Individuals can get indirect transmission of the disease when they come into contact with polluted materials or environments. People have the potential to become infected with Brucella bacteria if they come into contact with surfaces or things that have been contaminated with the bacterium. When reducing the risk of indirect transmission, it is necessary to implement the required sanitation and hygiene systems (Sarigüzel et al., 2011; Sánchez-Jiménez et al., 2020).

Although brucellosis is not often passed from mother to child, an infected woman can pass the disease to an unborn child while pregnant. This highlights the significance of prenatal treatment and monitoring in pregnant individuals with brucellosis (Schelling et al., 2003). For this reason, it is essential to gain a full understanding of the several modes of transmission to impede the spread of the disease. For the purpose of properly controlling this zoonotic sickness, it is essential to put into action effective preventive measures, such as enhancing public health knowledge and administering immunization to cattle.

Risk Factors

Risk factors are fundamental qualities that exist at the biological, psychological, family, communal, or cultural level and are associated with a higher likelihood of undesired results. These variables can influence the probability of negative consequences.

In humans

The disease that is commonly referred to as brucellosis is observed in every region of the world and is mandated to be reported in most countries. Regardless of the specific features that individuals possess, it has an impact on people of all ages and genders. Raw milk and its products, such as fresh cheese, are the primary substances responsible for the majority of cases that occur in the general population with this condition. The vast majority of these instances are accounted for by-products derived from sheep and goat populations. Because it is considered an occupational hazard, those who work in the cattle business are also at risk of contracting sickness (Schroeder and Coandton, 1917). This is because the disease occurs in cattle. Individuals who work in occupations that involve animals and who come into contact with blood, placenta, fetuses, and uterine fluids are at a greater risk of developing the disease. This is because these individuals are more likely to come into contact with these substances. In most cases, those who work in laboratories are affected by this form of transmission; however, it is also possible for individuals who engage in agriculture, meat processing, hunting, and veterinary care to be affected (Shamo’on and Izzat, 1999; Shenoy et al., 2016). The species known as Brucella melitensis is responsible for the majority of brucellosis cases that occur in humans all over the world. This is partly due to the challenges encountered while attempting to vaccinate goats and sheep that are allowed to roam throughout their natural habitat (Shepherd, 1991; Staszkiewicz et al., 1991; Sobsey et al., 2006; Sofian et al., 2008; Sousa et al., 2019).

In Animals

Many factors, including breed, physiological state, age, genetic resistance, sex, concurrent infections, stress, immune status, and body condition score, define animal-level risk variables. Several studies conducted by science have shown that different breeds of cattle show different degrees of sensitivity to bovine brucellosis. Genetically altered animals might show more hunger and limited access to cover, thereby increasing their susceptibility to disease. Comprehensive studies conducted in wealthy and underdeveloped nations have shown that the age of the animal is an important risk factor. The length of time people spend exposed to something increases as they age. Although they can get infected, symptoms do not appear until they are adults. Gender-related traits can affect individual behavioral patterns and the manner in which management is performed (Sung et al., 2012; Sun et al., 2020).

Male animals could exhibit more social interactions with other herds during the breeding season, thereby raising the possibility of disease spread. Lack of protein, minerals, and vitamins in the animal’s diet may lead to inadequate nutrition that will lessen the animal’s resistance to bovine brucellosis (Terefe et al., 2017). The size of herds, cattle management, and housing practices, interactions between animals and wildlife, introduction of new cattle, herd movement and trade, as well as past cases of bovine brucellosis in both animal and human populations resulting from transmission inside households, are risk factors that help spread bovine brucellosis. Adopting a nomadic lifestyle and interacting with a herd of animals and having a larger group size greatly increases the possibility of bovine brucellosis contact (Thimm, 2013; Tian et al., 2019). Potential risk factors include the frequency of the disease in the area or host animal, the concentration of wildlife within the group, and past M. bovis occurrences in wildlife populations following a direct link or shared living space with household animals (Tripp and Sawchuk, 2011; Traxler et al., 2013). This study emphasizes the need to increase knowledge about disease transmission and risk factors among those who directly interact with animals, mainly cattle owners. The need to avoid unpasteurized dairy products is also underlined, to lower the spread of this usually underappreciated zoonotic disease, thereby affecting the condition of a given area.

Public Health Importance

Brucellosis causes substantial economic losses in animals because it impairs fertility, decreases milk production, and results in frequent miscarriages in herds (Tulu, 2022). Human brucellosis is the most widespread zoonotic disease worldwide. Annually, around 500,000 new cases are diagnosed annually on a global scale (Ulu Kilic et al., 2013). Moreover, this condition is regarded as an occupational affliction that predominantly impacts veterinarians, farmers, stock inspectors, abattoir workers, laboratory specialists, butchers, and hunters. Given its significant infectivity via aerosol transmission, Brucella, particularly B. melitensis, could be considered a potential biological weapon (Valdes and Valdes, 2022). It has been approximated that a mere 10–100 organisms of B. melitensis are sufficient to induce aerosol infection in humans (Verraes et al., 2015).

Brucella abortus is an extremely pathogenic zoonotic bacterium that can cause severe sickness in humans. It is classified as an invasive species of Brucella in humans. Occupational exposure occurs when individuals come into contact with infected livestock or their tissues. Brucella infection is a frequently occurring form of infection acquired in laboratory settings. Humans can also acquire this illness by ingesting unpasteurized milk or milk products. The value is 84. Vaccination against strain 19 B. abortus might be harmful to individuals if not treated carefully (Williams and McKusick, 1954; Wardhan, 2020). Adverse events have been documented in RB51 vaccination, although it appears to be comparatively less risky than strain 19 (Xin et al., 2013). The occurrence of Brucella infection in people is linked to the prevalence of the virus in cattle. Brucellosis is a prevalent illness affecting both animals and humans in underdeveloped nations, resulting in a significant number of documented cases annually.

Economic Impact

Bovine brucellosis causes significant financial losses for the dairy business, notwithstanding the scarcity of comprehensive economic studies on this matter. Some researchers use terminology like economic impact, loss, and cost of brucellosis flexibly and interchangeably. The economic impact encompasses both direct expenditures, such as decreased milk production and higher mortality, and supplementary expenses, such as those related to vaccination and culling. The immediate repercussions can be categorized into two groups: conspicuous outcomes, such as abortion and repeated breeding, and subtle outcomes, such as diminished fertility. In addition, additional costs are associated with treatment and immunization, as well as the financial impact of selling animals at a reduced price due to distress (Yagupsky et al., 2019). Losses are specific elements that lead to a decrease in benefits, such as a decrease in milk production, weight gain, fertility, cost of replacing animals, and mortality. Costs, on the other hand, include expenses related to the treatment and control of the disease, such as the execution of measures to prevent the spread of the disease, vaccination, travel limitations, disease surveillance, and research. Many economic estimates have failed to take into account the financial impacts of forced selling, the loss of income due to terminated pregnancies in animals, the decrease in labor days spent on animal treatment, the costs of antiseptics and detergents, the expenses of transporting animals for treatment and the costs of diagnostic procedures.

Many research studies heavily depend on limited epidemiology data and utilize assumptions acquired from the country or other sources to construct economic projections (Yohannes et al., 2012; Yasmin and Lone, 2015). There is a lack of research that accurately measures the economic effects of the disease using reliable epidemiological data gathered from a randomly chosen population. The diverse estimations of economic impact, cost, and loss result from the absence of standardized assessment methodologies and the dependence on distinct environmental elements (Yoo, 2010; Young, 2020).

Treatment

The treatment of brucellosis in animals is typically ineffective because the organisms responsible for the disease are located inside the cells, allowing the bacteria to remain and multiply (Yumuk and O’Callaghan, 2012). Brucella infection is typically introduced to the herd by diseased cattle, as well as through the transfer of the disease from infected bulls’ semen or contaminated items (fomites) (Zhang et al., 2018). The main principles of treatment include the utilization of combination regimens and prolonged medication duration. Efforts to promptly address antibiotic treatment are essential although it is not effective in animals. More precisely, antibiotics are administered to farm, pet, and zoo animals to treat bovine brucellosis. It is crucial to acknowledge that clinical improvement can occur even in the absence of a bacteriological cure (Zheludkov and Tsirelson, 2010). Even animals that initially exhibit a positive response may later experience relapse, particularly if the treatment provided is insufficient, such as the use of a single medication or the use of an extremely short duration of treatment. The potential for the emergence of drug resistance, the danger of pathogen transmission, and potential harm to persons, particularly in the context of draining wounds or respiratory tract infection. The administration of vaccines to calves or heifers is the most effective approach for managing Brucella in regions with high disease prevalence Torgerson et al. (2018). Cattle that have been recently introduced should be both free from Brucella and sourced from disease-free areas. Before introducing additional animals into the group, it is imperative to isolate and test them to determine whether they are carrying the Brucella infection Yagupsky et al. (2019).

Prevention and Control

The primary objective of brucellosis management in animals is to mitigate its impact on human health and minimize economic losses. The fundamental tenets of animal control methods, as elucidated in a prior investigation, encompass the examination, segregation, and/or euthanasia of animals, the limitation of their mobility, and the administration of immunizations (Collett et al., 2020). Vaccination is the most effective approach for preventing and managing brucellosis (Nyerere et al., 2020). The vaccinations for animal brucellosis include live strains of B. abortus (specifically strain 19 and strain RB5), B. suis S-2, rough B. melitensis strain M111, and weakened strains of B. melitensis strain Rev.1. Vaccines are accessible and can efficiently prevent infections caused by B. abortus and B. melitensis. The dominance of B. melitensis strains Rev.1 and B. abortus strain 19 over all other strains has been demonstrated. Despite thorough and careful research efforts, no vaccine has received approval for the protection of human brucellosis (Schurig et al., 2002; Hou et al., 2019; Heidary et al., 2022).

To reduce the risk of transmitting brucellosis to humans, a variety of effective techniques are recommended. The preventive measures include pasteurization of dairy products, thorough cooking of meat, cautious handling of aborted biological materials and newborns, wearing protective clothing in high-risk areas, exercising extreme care when administering S19, RB51, or Rev 1 Brucella vaccines, and raising community awareness about these preventive factors. Suggestions for the control and prevention of brucellosis include implementing stricter laws for food safety, following proper hygiene practices, and establishing thorough surveillance systems. Both healthcare providers and the public need to have a higher level of awareness and education (Tabar and Jafari, 2014; Pérez-Sancho et al., 2015).

To evaluate the existing disease burden, it is imperative to promptly identify and gather accurate information regarding prospective carriers. Efficient national surveillance and regulation necessitate cooperation among many federal ministries and entities. To evaluate the effects of zoonotic diseases on society and the economy, one can analyze measures such as disability-adjusted life years to compare the overall illness burden (Torgerson et al., 2018). Subsequently, these data can be utilized to generate informed choices regarding the administration of brucellosis initiatives. The historical occurrences of quarantine as a result of brucellosis, such as the situation where British soldiers were exposed to milk infected with Brucella, emphasize the significance of efficiently dealing with the existence and spread of the illness (Qureshi et al., 2023). The disease can be completely eliminated by adopting quarantine measures and eliminating diseased animals. Strategies to mitigate the risk of transmission of brucellosis through milk and other dairy products involve instituting stringent heat treatment procedures before consumption and improving the safety protocols of dairy supply chains.

Individuals who come into contact with Brucella, such as veterinarians, laboratory workers, and those who handle infected animals, should use the necessary personal protective equipment and receive sufficient training (Abd El-Hameed et al., 2012).

Conclusion

Bovine Brucellosis is a prevalent disease that has a substantial impact on the economics of the cattle industry. Although it is the second most dangerous zoonotic disease worldwide, behind rabies, this sickness is frequently disregarded, despite its substantial effects on public health and the economy. However, the implementation of milk pasteurization, diagnostic testing, and the elimination of contaminated cattle has resulted in a substantial reduction in the prevalence of bovine brucellosis in industrialized countries. Efficient management of bovine brucellosis is crucial because it adversely affects the productivity of affected animals and poses a potential threat to human health.

Funding

The APC was generously funded by Airlangga University, a prestigious institution based in Surabaya, Indonesia, known for its academic excellence and commitment to fostering innovative research.

Authors Contribution

All authors contributed to the data analysis, the drafting and revision of the manuscript, and agreed to take responsibility for all aspects of this research.

Conflict of interest

All authors declare that they have no conflicts of interest.

Data Availability

The data for this review article was accessible to the primary author, and the details regarding the corresponding author can be made available upon special request.