Brucellosis: Current Status Of The Disease And Future Perspectives

Brucellosis is an infectious zoonotic disease of domestic animals, as well as humans, globally [1–3]. Various domestic animals such as camels, goats, sheep, cows as well as humans are affected by brucellosis. It is caused by the Brucella species such as Brucella melitensis in small ruminants, Brucella abortus in cattle, and Brucella canis in dogs [4–5] Brucella species are Gram-negative, small coccobacillus, slow-growing and intracellular bacteria that are capable of surviving and multiplying within macrophages, dendritic cells, placental trophoblasts, and epithelial cells. Brucella species can survive under extreme conditions of temperature, humidity, pH, and persist in frozen and aborted materials for longer durations [6].

Although several countries are affected by brucellosis, it is still a neglected disease and no official program for surveillance and eradication of animal brucellosis has been proposed [7]. In many developing countries of Africa, Asia, Central, and South America, clinical disease is recorded among different animals [8, 9]. The impact of brucellosis on human health is a major issue [10]. In humans, it causes fever, nausea, muscular pain, abdominal pain, sweating, weakness, decreased appetite, weight loss, and liver inflammation [11]. This disease in domestic animals poses a threat to free animal movement and trade of animals and various animal products and causes huge economic losses. It also leads to economic burden due to decreased milk production, breeding failure, and abortion in the domestic animals infected with Brucella spp. [12].

The development of DNA markers and molecular biology techniques are important tools for genomics-based studies in animal biotechnology [13]. It has led to genetic improvement and markers now help in the selection of best quality breeds of animals. Over the last 20 years, DNA markers have resulted in tremendous genetic improvement in farm animals. Recent developments in molecular biology techniques have revealed genetic polymorphisms in DNA sequences, which have been used extensively as markers for assessing the genetic basis of the phenotypic variations in animals. The genetic markers indicative of changes at the DNA level are called molecular markers [14]. The PCR has become an important tool for molecular DNA studies, including the detection of DNA polymorphism (fingerprinting), analysis of genotyping, and genome mapping. Along with microbial techniques, 16S rRNA gene sequencing and RAPD methods are very essential in molecular diagnosis. Using an array of random single RAPD primers, it is possible to determine the polymorphism that can be used in DNA analysis [15].

Improvement of the livestock industry and the use of improved prevention measures to control brucellosis, require an understanding of the disease risk and the prevalence of infection in the human population and animal hosts. Epidemiological studies of brucellosis in humans as well as domestic animals globally is urgently needed to effectively control this disease [16–21]. The objective of this work are to describes the current status of brucellosis and the risk factors of the disease in animals and the human population. In addition, there is a further discussion of the various issues related to the control and prevention of brucellosis in domestic animals and humans.

The disease brucellosis has existed since time immemorial. The analysis of the skeleton of Australopithecus africanus from the late Pliocene era revealed the effect of brucellosis in human ancestors [15]. Furthermore, DNA analysis and pathological findings from human skeletal remains and buried cheese remains indicated the presence of brucellosis in some countries in the Middle East and Europe in 79 A. D. [15, 16]. The earliest record of this disease was in 1859, and it mentioned that animal abortions were most probably due to brucellosis [1]. However, in the 1880s, the causative agent of this disease, Brucella melitensis, was isolated and identified. The term brucellosis was named after Sir David Bruce who isolated and characterized the infectious agent from a soldier in Malta in 1886. This disease was responsible for inflicting severe mortality and morbidity in British military personnel in Malta and hence was also well-known as Malta or Mediterranean fever [6]. It was also known as Bang’s disease after the isolation of the causative agent Brucella abortus in 1897 by Danish veterinarian Bernhard Bang. Due to the “wave-like” characteristic of the fever, which rises and falls over several weeks in patients, it is also known as “undulant fever”. Other names for brucellosis include Gibraltar fever, Rock fever, Cyprus fever, and typhomalarial fever in humans and animals.

Although Brucellosis has been very well controlled in many developed countries, it is still a major concern in Asia including Middle Eastern countries, Africa, Mediterranean countries and South America. Human brucellosis has re-emerged in China since 1990, due to the drastic growth of animal husbandry which increases the probability of human infection [3]. Brucellosis is still a major concern in the Indian subcontinent. India has one of the largest bovine populations in the world, which is responsible for the continued exposure of workers in milk industry to these animals and hence there are high incidences of brucellosis in humans [17]. A seroprevalence study in India indicated brucellosis seroprevalence ranging from 2% to 18% in suspected patients [17]. Several countries which were endemic such as Malta, France, Ireland, Israel have been successful in eradicating the disease. Studies in various countries, have indicated that B. melitensis is the main cause of human brucellosis, while infection with B. abortus is less frequent [22]. Domestic animals are a natural reservoir of Brucella spp. and animal-to-human transmission occurs through the consumption of infected meat and milk. Serological data for the prevalence of brucellosis in various provinces in Saudi Arabia is still not available [22, 23]. The incidence of the disease had been reported from the Northern, Southern, and Central regions of Saudi Arabia [24, 25]. A seroprevalence study by Ageely and colleagues in the Jazan province of Saudi Arabia revealed that the prevalence of human brucellosis was higher in patients ≥ 40 years old (20 %) as compared to the population < 40 years (12 %). Seroprevalence was higher in the rural population (39.3%) than in the urban population (4.6%), and significantly higher in the Saudi population (14.5%) compared to the non-Saudi population (3.0%). However, the prevalence was much higher in males (16.4%) than females (7.1%) [16]. Brucella spp. infection in humans is mainly through the consumption of raw milk and meat products derived from infected goats or camels. It has also been observed that laboratory workers, hospital staff, and veterinarians are more prone to brucellosis [23, 26, 27]. Brucellosis is caused by bacteria belonging to genus Brucella in humans and several other animals, including goat, cows, buffaloes, sheep, pigs, camels and reindeer [1, 28].

Brucella spp. are Gram-negative cocci or small rods, non-motile, non-encapsulated, non-flagellated, nonspore forming aerobic microbes. It has the capability to invade, epithelial cells, macrophages, dendritic cells and placental trophoblasts [7].

Genus Brucella belongs to the alpha-2 subdivision of class Proteobacteria and 10 different species of Brucella based on the host specificity that has been reported. These species are B. melitensis (goats), B. abortus (cattle), B. ovis (sheep), B. canis (dogs), B. neotomae (desert woodrats), B. suis (swine), B. pinnipedia (seal), B. microti (voles), B. cetacea (cetacean), and B. inopinata (unknown) [29]. Among the known 10 species of Brucella only B. melitensis, B. abortus, B. canis and B. suis, have been found to cause infection in humans. B. ovis and B. neotomae are not pathogenic to humans. Most of the human infection cases globally are caused by B. melitensis [30]. B. melitensis and B. suis are more infectious and virulent in humans compared to B. canis or B. abortus [31]. B. melitensis, B. suis and B. abortus are known to have 3, 5, and 7 subtypes, respectively [32, 33]. Sequencing the genome of Brucella species revealed a sequence homology of more than 99% among the species [29, 34].

Most Brucella species possess smooth lipopolysaccharide (SLPS) in the outer cell wall, while B. canis and B. ovis have rough lipopolysaccharide (RLPS) and protein antigens [35]. SLPS contains an immunogenic O-polysaccharide, defined as a homopolymer of 4, 6-dideoxy-4-formamide-α-D mannose, which is connected by glycosidic linkages. Brucella species that possess smooth lipopolysaccharide, particularly B. melitensis, are known to undergo dramatic structural variations during growth, and more often change to rough form (R) and occasionally to mucoid form (M). During the change in morphology to rough form (R), bacterial colonies appear to be less transparent, with a more granular surface. During the change in morphology to mucoid form (M), the bacterial cells appear to have a gelatinous texture and their color changes from white to brown in reflected light. An intermediate form (I) has also been observed during the change in morphology from smooth (S) to rough form (R). It had been shown that changes in the bacterial cell morphology are associated with marked differences in virulence, pathogenicity and serological properties of Brucella spp. [36].

The epidemiology of brucellosis has dramatically changed over the past few years due to improvement in hygiene, socio-economic conditions and an increase in international travel. Incidence of human brucellosis has been reported for the first time from the regions of central Asia and in some countries. Particularly in Middle East countries, there is a drastic increase in the incidences of brucellosis in humans [30].

Brucellosis affects domestic as well as wild animals. It has been reported to occur worldwide wherever animals are raised [37]. Although some industrialized countries in Europe and America have been able to eradicate brucellosis in domestic animals through intensive control schemes, the disease is still a severe problem in several developing countries [38].

B. melitensis is the most pathogenic species and comprises three biovars. Biovars 1 and 3 have been detected most often in domestic animals in the Middle-East, Mediterranean and Latin American countries [39, 40]. Brucellosis is considered a major barrier to the free trade of domestic animals and various animal products and is responsible for significant economic losses due to abortion in domestic animals [41].

Various factors such as environmental factors and the host biology can affect the occurrence and prevalence of brucellosis. Some of these factors include the age of the animals, herd size, sanitary conditions of animal farms and climatic conditions [24, 42] It has also been observed that sexually mature animals are more prone to the infection and bacteria mainly localize in the reproductive organs, particularly in pregnant animals. Besides, Brucella spp. may also be localized in the mammary glands [43].

Many people in poor African countries such as Ethiopia are dependent on livestock for their livelihood which leads to their close association with the domestic animals, increasing the risk of infection through Brucella spp. [42, 44]. Cases of brucellosis are very high in rural areas as farmers live closely with their domestic animals and more often consume unpasteurized milk products [24]. In countries like Ethiopia, the habit of consuming raw milk, improper handling of an aborted fetus and reproductive excretions are responsible for the transmission of zoonotic diseases like brucellosis to humans [45].

In many other countries, risk factors for infection with Brucella include consumption of contaminated animal products such as milk and meat, handling of infected animals, traveling to an endemic area and mishandling cultures of Brucella sp. in laboratories and diagnostic centers. Veterinarians, dairy workers, and slaughterhouse workers are more susceptible to infection with Brucella [8]. Recent studies showed a poor community’s knowledge of brucellosis and the risk associated with brucellosis among people living adjacent to Awash National Park in Ethiopia [46]. Hence, there is an utmost need to create awareness about the disease, improve knowledge, attitudes and practices among livestock owners, which would further lead to a significant reduction in the transmission of the bacteria from animals to humans in the disease-prone areas [41].

Brucella melitensis has been found to be sexually transmitted in sheep and goats. The transmission of brucellosis is facilitated by the intermingling of animal herds that belong to different owners and by the procurement of cattle from sources that are not properly screened for the disease [2]. Moreover, using common male breeding stock also increases the risk of disease transmission among domestic animals. Other factors that can promote the transfer of infection include the intermingling of animals during grazing, the crowding of animals in farms, marketplaces or animal fairs [1]. Following an abortion in domestic animals, animal farms can become contaminated with Brucella spp. and other animals on the farm may acquire the disease by ingestion, inhalation, skin contamination or udder inoculation. Sometimes, pooled colostrums used for feeding newborns can also play a role in the transmission of the disease. Artificial insemination may also promote the transmission of the Brucella spp. to healthy animals.

In humans, brucellosis is caused by B. melitensis, B. abortus and B. suis which are transmitted by an infected goat, pigs, sheep or cows to healthy humans [47]. Besides, domestic and wild animals are also infected with Brucella sp. and can act as reservoirs of bacteria that can be transmitted to both humans and domestic animals [41]. Exposure of humans to infected domestic animals or the consumption of milk or meat products derived from infected animals enhances the risk of acquiring brucellosis [8]. The transmission of brucellosis may also occur through blood transfusion or organ transplantation. Some people such as farmers, farm laborers, animal attendants, shepherds, and veterinarians are at a very high risk of acquiring the infection. These workers are always at a very high risk of infection with Brucella spp. due to direct contact with infected animals or constant exposure to the contaminated environments. The main source of Brucella infection in the urban population is usually contaminated food, milk or dairy products derived from infected animals [20, 47].

In humans, there are three stages of brucellosis i.e. acute, subacute or chronic phase and the incubation period is two to three weeks and sometimes several months. The predominant symptoms of brucellosis in humans include intermittent fever, headache, backache, weakness, weight loss, anorexia and mental depression [52, 53]. During the chronic phase, knee joints are also affected [54]. Complications may occur in the gastrointestinal, cardiovascular, pulmonary, lymphatic, and nervous systems [55, 56]. The effect of Brucella infection on the nervous system causes neuro-brucellosis, which is characterized by fever, headache, psychosis, seizures, behavioral changes, and spastic paresis [57].

It has always been difficult to clinically diagnose brucellosis in animals as well as in humans [13]. Presently, the serological method, culture-based method, and molecular techniques are employed to detect Brucella infection in animals and humans [58]. Diagnostic tests are based on the detection of the causative agent Brucella sp., the detection of antibodies in the serum and allergic reactions [59].

Several serological tests are currently being employed for qualitative and quantitative detection of specific immunoglobulins of Brucella organism-specific antibody titer in the infected animals. Serological tests such as Rose Bengal Plate test (RBPT), Complement Fixation Test (CFT), Serum Agglutination Test (SAT), and Enzyme-Linked Immunosorbent Assay (ELISA) are routinely used for diagnosis of brucellosis in animals and human [60]. Milk Ring Test (MRT) is employed for the detection of Brucella organisms in infected animals [62].

Currently, molecular biology techniques are being used extensively to identify the causative agents, as these techniques are less time-consuming, have high specificity and sensitivity to detect microorganisms [60, 68]. In addition, restriction endonuclease and hybridization have been used extensively for decades for Brucella detection [14, 69].

PCR is very sensitive, highly specific, rapid, and easily amenable for high-throughput screening. It is also more suitable for the detection of slow-growing bacteria such as Brucella [70]. Due to its very high sensitivity, the PCR based method has the capability to detect tiny amounts of bacteria in clinical samples. It has also been demonstrated that the PCR technique is able to detect dead microbes in clinical samples, hence reducing the need for proper sample preservation before analysis [71]. This method of detecting Brucella infection is highly reproducible and very reliable, however, care should be taken to avoid contamination during analysis in the laboratory [72].

PCR-based protocols for the detection of Brucella spp. in clinical samples have been designed and developed. These methods are based on the amplification of gene BCSP31 which is highly conserved among Brucella spp., or the amplification of the 16S rRNA gene [72]. This approach is useful for screening when biovar or species designation is not critical. In order to distinguish Brucella species or biovar, PCR protocols have been developed based on gene loci, which vary among biovars and species. However, such gene targets are not common in Brucella, as the genus is unusually homogeneous and conserved. While few deletions and rearrangements have been found within a biovar or species, most of the differences at genetic levels consist of single nucleotide polymorphisms [69]. Differential PCR based methods are of great value for epidemiological studies, disease monitoring, and species-specific eradication program. The differential PCR protocols can be denoted as species-specific and genus specific. In the case of speciesspecific methods, three approaches have been reported (i) protocols designed with highly specific primers and stringent assay conditions; (ii) protocols developed with low-specific primers and less stringent PCR conditions, and (iii) protocols designed with random primers under very flexible assay conditions [73, 77].

Brucellosis is an infectious disease that has been controlled and eradicated in some countries in the world [3]. In sub-Saharan Africa, animal health services have been substantially deteriorated over the last 20 years due to various factors such as reduction in government budgets, especially the funds required to control brucellosis [81] Hence, various programs that require the use of disease prevention measures, information exchange, and coordinated surveillance, are not properly implemented in many sub-Saharan countries [2, 18, 42, 82].

The primary objectives for the control and prevention of brucellosis are centered on the economic impacts of the disease and its public health consequences [73]. Control activities mainly reported by countries include surveillance, controlling the movement of domestic animals, treatment of meat and milk products and animal vaccination, [3, 42, 84]. In Mozambique, the control of brucellosis was well organized by using the S19 vaccine in cattle until 1980. A strain 19 vaccine produced by the Underreport Veterinary Institute in South Africa containing viable Brucella cells was used. The vaccine was administered subcutaneously to heifer calves at four to eight months of age. However, recently some private farmers have been using S19 vaccination in adult cows. Also, surveillance and movement control were also implemented at a very low level, resulting in a drastic increase in cases of brucellosis. Brucella abortus vaccine, strain RB-51, live culture, licensed in 1996, has been extensively used in USA to eradicate brucellosis [73–77].

Antibodies against Brucella cell wall O-polysaccharide (OPS) component of smooth lipopolysaccharide is known to confer protective efficacy to the currently used vaccines. However, the detection of these antibodies is also used in diagnosis of brucellosis in animals, and so it becomes very difficult to differentiate vaccinated animals from infected animals and this hampers the effort to control the disease [75]. In order to sort out this issue, it has been proposed that combining anti-Brucella OPS antibody response with the induction of cell-mediated response would provide highly effective protection against brucellosis which can be achieved by the conjugation of O-polysaccharide (OPS) to a highly immunogenic Brucella protein. Such type of glycoconjugate vaccine would be more effective in protecting animals and humans against brucellosis and would not interfere with the diagnostic testing for Brucella infection [75].