Open access peer-reviewed chapter

Control of Animal Brucellosis — The Most Effective Tool to Prevent Human Brucellosis

Written By

Marta Pérez-Sancho, Teresa García-Seco, Lucas Domínguez and Julio Álvarez

Submitted: December 19th, 2014 Reviewed: July 14th, 2015 Published: November 19th, 2015

DOI: 10.5772/61222

Chapter metrics overview

2,812 Chapter Downloads

View Full Metrics


The World Health Organization classifies brucellosis as one of the seven neglected endemic zoonosis which contribute to the perpetuation of poverty in developing countries. Although most of the developed countries are free from this important zoonosis, brucellosis has still a widespread distribution in the Mediterranean region, the Middle East, Central Asia, and parts of Latin America, making it a global problem. Nearly half a million of new cases of human brucellosis are reported each year around the world, in which animals (or products of animal origin) are the most likely source of infection. Brucella melitensis, the main etiologic agent of small ruminant brucellosis, is the most prevalent specie involved in cases of human disease in most parts of the world. Additionally, Brucella abortus (main responsible of bovine brucellosis) and Brucella suis (the most common etiological agent of porcine brucellosis) are often associated with human brucellosis. In animal production, brucellosis has a strong economic impact due not only to its direct consequences (e.g., reproductive failures) but also to indirect loses (e.g., trade restrictions). The problem of brucellosis could be considered a clear example of the need for a “One World, One Health” strategy, given that the only approach to achieve its control and subsequent eradication is the cooperation between public and animal health authorities. The prevention of human brucellosis cannot be achieved without the control of the disease in the animals, as exemplified by the impact that the early measures adopted in the beginning of the 20th century forbidding the consumption of goat milk had on the prevalence of the disease in the British soldiers in Malta. When the prevalence of the disease in the animal population is high or when eradication cannot be achieved due to other factors (e.g., lack of economic resources), its control in livestock must be the first objective. When deciding the optimal approach to tackle the disease, the prevalence of animal brucellosis is not the only parameter to consider by the decision makers since other epidemiological and economic aspects should be considered in order to implement the most adequate control strategy in each region. Cooperation between all stakeholders involved is a cornerstone in the success of any control strategy. Strict biosafety and management measures, vaccination, and test-and-slaughter strategy are recognized as the most effective strategies to control this pathology in livestock. The adequate combination of these measures depends on several factors that will determine the success of the eradication efforts. The present chapter will review the abovementioned measures for the control and eradication of brucellosis in livestock, focusing on the advantages and drawbacks of the diagnosis tools and immunization strategies currently available and evaluating new approaches based on the advance on the knowledge of different aspects of this disease and its etiological agents.

1. Introduction

Animal brucellosis is one of the most important challenges faced by animal health authorities and producers worldwide due to the large number of host species that can be affected, limitations of the currently available diagnostic and prophylactic tools, and complex epidemiology. Yet, control and eventual eradication of animal brucellosis is the only way to ultimately win the battle against human brucellosis. Although the present chapter is focused on the current approaches for the control of B. melitensis in small ruminants (small ruminant brucellosis, SRB), B. abortus in cattle (bovine brucellosis, BB), and B. suis (biovars 1, 2, and 3) in swine (porcine brucellosis, PB), the complex and dynamic nature of the epidemiology of animal brucellosis must be borne in mind when analyzing a given epidemiological setting since sometimes certain Brucella species can be found in host species other than their preferred ones (for example, B. abortus may be the etiological agent of brucellosis in sheep [1, 2]). A perfect example of this complex situation is shown by the increasing importance of B. melitensis in cattle in some Mediterranean countries, e.g., Saudi Arabia, Egypt, and Kuwait [3-5].

The need for a control of animal brucellosis has been a major concern since the first report describing the implication of animals in the epidemiology of the disease in 1905 [6]. Although brucellosis had been known for centuries as a chronic recurrent febrile disease mostly present in the Mediterranean region, it was not until the end of the 19th century when it was recognized as a dramatic disease wreaking havoc among the British army stationed in Malta. In 1887, Sir David Bruce (a British surgeon whose surname would later give name to the genus) isolated the etiological agent (firstly named as Micrococcus melitensis) of the infectious disease that was affecting an increasing number of soldiers in the island [7]. In 1904, the great concern raised by the impact of brucellosis in Malta contributed to the constitution of the “Mediterranean Fever Commission (MFC),” with Sir Bruce as the president of the organization. One of the main aims of the commission was to identify the sources of infection of the disease, an objective that was finally achieved by serendipity [8]: Sir Themistocles Zammit, a Maltese doctor member of MFC, included goats for experiments due to the temporary shortage of monkeys, traditionally used for in vivo studies. Surprisingly, agglutinins and bacteria were detected in the blood and milk of infected goats, thus suggesting these small ruminants were susceptible to the disease and a potential source of infection [6]. The ban on the consumption of Maltese goat milk among British soldiers was the first step for the control of this dramatic zoonosis in the island [9], one of the first preventive measures to control the transmission of Brucella from animals to humans.

Brucellosis can be considered a paradigm of the need for a “One World, One Health” strategy given that the only approach to achieve the control and subsequent eradication of this zoonotic disease is the cooperation between the industry, producers, and public and animal health authorities [10]. Human-to-human transmission, although possible and occasionally reported due to transplantation, sexual contact, and lactation [11-14], has an insignificant impact on the epidemiology of the disease since humans are traditionally considered dead-end hosts [15]. The major sources of infection for human are therefore infected animals, not only due to direct contact but also - and most importantly - through the consumption of raw dairy products [3]. Traditionally, the main etiological agent of human brucellosis is B. melitensis, although a relevant role of B. abortus and B. suis (mainly biovars 1 and 3) has also been described. Although approximately 500,000 new human cases of brucellosis are reported every year around the world [16, 17], underdiagnosis/underreporting of human brucellosis is a major issue in many regions [18, 19]. The control of the disease in humans is impaired by the lack of available vaccines [20], thus leaving the control of animal brucellosis as the most effective strategy to prevent human infection [19, 21, 22]. In fact, surveillance systems for human brucellosis can act as sentinel tools of the situation of the disease in animals since the occurrence of human cases can be one of the first indicators of the presence of disease in the animal population [23], and likewise, a decreasing trend in the number of human cases may suggest that brucellosis control campaigns are effective [24, 25].

However, and despite its crucial importance from the public health perspective, the justification for the control of animal brucellosis rests not only on its zoonotic nature but also in the severe losses that its presence entails. Economic costs derived from the presence of Brucella infection in animals are derived from the direct consequences of the disease (abortion, infertility, reduction of milk production, orchitis, epididymitis, etc.) and the indirect losses (replacement of reactors, costs associated to control/eradications programs, movement restrictions, trade limitations, etc. [23, 26]). In addition, brucellosis has been recognized as a neglected zoonotic disease that contributes to the perpetuation of the poverty in endemic regions of low-income countries, compromising their economic development [27, 28]. Moreover, costs due to human brucellosis, as the investments on treatments, prevention of the disease, and loss of productivity are other overheads attributable to animal brucellosis.


2. Control and eradication strategies for animal brucellosis

Despite the huge efforts invested on the control of animal brucellosis, results have not always matched the expectations, particularly in the case of ovine and caprine brucellosis, in which control has proven to be more challenging than that of bovine brucellosis due to B. abortus. This situation may be the consequence of the combined effect of several factors, including those inherent to the disease regardless of the etiological agent/infected host [existence of a prolonged latent period often associated with lack of serological responses [29] and limited sensitivity of some diagnostic tests in certain epidemiological situations [30, 31] and also other factors associated with the etiological agent (environmental resistance of B. melitensis and B. abortus [32]) and the host (traditional farming practices as communal pastures and transhumance practices, typical of small ruminants [33, 34]).

Still, three major strategies have been demonstrated as effective tools to control brucellosis in domestic animals when used in combination:

  1. Strict biosecurity at the farm level

  2. Test and slaughter programs

  3. Immunization of the susceptible population

The sole implementation of one of these measures is however much less effective since optimal results are obtained when at least two of them are applied jointly. Still, the best strategy will depend on the epidemiological situation in a given setting, the availability of resources, etc. [26]. Moreover, in addition to these “classical” strategies, other complementary tools should be considered to ensure the success of the program (animal identification, animal movement control, economic compensations, etc.) [33].

The present chapter will review the tools currently available to achieve the control and eradication of brucellosis in livestock (bovine, porcine, and small ruminant brucellosis), focusing on the advantages and drawbacks of the diagnostic tools and the immunization strategies the two main pillars in which control programs are based. New approaches based on the advance in the knowledge of different aspects of the disease and of their etiological agents will also be reviewed. Finally, the factors that should be considered when selecting the most suitable strategy for control of small ruminant, bovine, and porcine brucellosis and that often determine the success of the control/eradication efforts will be discussed.

2.1. Management and biosecurity

Management and hygienic measures against Brucella infection must be focused in diminishing the possibility of contact with viable Brucella, including both infected animals and contaminated environments.

The most frequent routes of entry of Brucella in a free farm are the following:

  • Purchase of infected animals that can shed the bacteria to the environment, therefore exposing susceptible individuals. In ruminants, up to 1010-1013 CFU/g of tissue and membranes of aborted fetus can be excreted during the clinical phase of the disease [35]. In swine, infected boar may excrete 104-105 CFU/ml of semen, thus turning venereal transmission one of the most important routes of infection for B. suis, particularly in brucellosis-free settings in which artificial insemination can constitute an important risk factor [32].

  • Contact with infected material, pastures, etc., due to the high environmental resistance of Brucella spp., which leads to its persistence outside the host for long periods, allowing a variety of transmission routes of Brucella (conjunctival, oral, and respiratory).

Thus, the use of appropriate biosecurity measures is of critical importance to prevent the entrance of the disease in a naïve epidemiological unit. These strategies include the implementation of quarantine before the introduction of new animals, the separation of animals with an unknown/uncertain status, the control of animal movements, the adequate management of replacement, the isolation of pregnant females before parturition (particularly primiparous animals), and the strict quality/sanitary control of semen. In case of artificial insemination, avoid or limit the contact between livestock and wildlife in environments where wild animals have been seen to be a source of infection [23, 29, 36, 37].

In infected settings, in addition to the biosecurity recommendations cited above, hygienic measures are essential to limit and control the bacterial load in the environment, decreasing the possibility of contact with viable Brucella spp., and should be systematically implemented. Removal of abortion products, full cleaning and disinfection of premises, elimination of infected manure, and incineration of infected material are some examples of measures to attain this objective.

Certain management/farming practices (traditionally used in small flocks from endemic region of low-income countries), such as nomadism, mixing animals from different origins at grazing, and use of shared pastures, may favor transmission of the bacteria, thus hindering the effectiveness of control strategies [38, 39].

2.2. Test and slaughter programs

The main aim of this approach is the early detection and removal of possible sources of infection (infectious animals), thus avoiding circulation of Brucella. Despite the effectiveness of the diagnostic strategy used, there is always a certain risk of having infected animals that may remain as silent carriers [40] maintaining the pathogen in the flock and, if there is a drop in the immunity of the herd, may lead to an abortion storm. This strategy is most useful in low-prevalence settings where economic resources and veterinary expertise are available for its support [41]. Test and slaughter strategies may also be useful for the management of outbreaks, particularly when numbers of animals make the implementation of stamping-out measures unfeasible [42]. Although the tests used for the detection of infection can be classified according to different criteria, this section is organized based on its ability to detect the pathogen (direct tests) or the immune response induced in the infected host (indirect tests that can be further subdivided on account of the immune response they target, humoral or cellular). In some cases, the only measure that achieves complete elimination of the bacteria on the flock is the stamping out followed by a thorough cleaning and disinfection and replacement with Brucella-free animals [43].

2.2.1. Indirect diagnostic tests

Most of these techniques (especially those using inactivated whole-cell suspensions of Brucella as antigens) were developed for the diagnosis of bovine brucellosis in the first place and were further adapted later for its application in small ruminants and swine considering that the principle of all techniques is the same regardless of the Brucella species/host: all the major tests are based on the detection of antibodies against the smooth lipopolysaccharide (S-LPS; the immunodominant antigen of smooth Brucella species: B. abortus, B. melitensis, and B. suis) [44]. The outer-polysaccharide chain (O-PS), the main antigen moiety of smooth Brucella LPS, shows a different proportion of α-1,2 and α-1,3 linkages dividing strains in two groups: A-strains or M-strains, depending on the quantitative distribution of A and M antigens in the smooth species. Thus, the suitability of Brucella antigens recovered from M-dominant strains (such as B. abortus biovar 1, typically used for rose bengal test (RBT), complement fixation test (CFT), and some ELISA techniques) for the diagnosis of brucellosis due A-dominant strains (e.g., B. melitensis biovar 1) has been questioned [45]. In fact, some authors have shown that RB and CFT may present an impaired diagnostic performance when used on small ruminants (particularly in goats) compared with that observed in cattle [46]. However, other studies have demonstrated that the use of B. abortus antigens for performing RB and other serological techniques allows an appropriate sensitivity for its use in B. melitensis diagnosis in small ruminants [47, 48]. A possible explanation for this finding would be the existence of common epitopes (C-antigen) present in the LPS of A- and M-dominant Brucella strains [49].

The accuracy of the diagnostic tests in the control/eradication programs of brucellosis is an essential component in the success of test and control strategies [50]. Although the diagnostic performance of most of the currently available diagnostic techniques has been demonstrated to be adequate, there are some epidemiological situations in which serological diagnosis may have some limitations. For example, silent carriers of the disease (e.g., infants infected congenitally in utero or by ingestion of contaminated colostrum/milk) may remain seronegative until a reproductive failure occurs [51]. In addition, positive results may not always be indicative of an active infection [52] since the occurrence of diagnostic interferences associated with exposure to other Gram-negative bacteria has been extensively demonstrated [53]. These microorganisms have LPS molecules similar to the Brucella-LPS in the outer membrane and may induce the production of cross-reacting antibodies, thus leading to false-positive results in the traditional serological techniques for brucellosis diagnosis. Different bacteria (including Escherichia coli O:116, E. coli O:157, and Vibrio cholerae O:1) have been recognized as a potential cause of this diagnostic interference, but Yersinia enterocolitica serotype O:9 is considered the main agent compromising the diagnostic specificity of serological tests [54]. This is a major challenge in the case of swine brucellosis [32, 55], especially when biovar 2 occurs, because its LPS antigenic structure is more similar to Y. enterocolitica than to other biovars of B. suis [56]. Most of these cross-reacting antibodies belong to the M isotype, and therefore the implementation of serological techniques based on the predominant detection of IgG1 is potentially more useful [53].

The diagnostic specificity of serological tests may also be compromised by the occurrence of false-positive reactions caused by antibodies induced by vaccine strains (mainly the smooth B. melitensis Rev. 1 and B. abortus S19, the most widely used strains for immunization against small ruminants and bovine brucellosis, respectively).

There is not a single serological test that can detect 100% of the infected population, and even used in combination may miss up to 30% of the infected animals using some serological tests [45], although its sensitivity at the herd level is much higher. In fact, in the frame of control and eradication programs, serological results are often interpreted at the herd level [46]; hence, the presence of one reactor involves the possible exposure to Brucella spp. of all animals of the flock that are therefore considered suspected and put under restriction measures until the infection is considered cleared in the herd/flock.

However, limitations in terms of the specificity of serological tests (such as those stated before) lead to the need of confirming the infection using other tools (epidemiological evidences of exposures and, preferably, isolation of the bacteria) at the herd level. Culture is considered the gold standard technique for the confirmation of the infection due to its high specificity [57], although this method is not free of drawbacks (see below).

The existence of international standards for diagnostic tests, vaccines requirements, reporting data systems, etc., is a cornerstone in the implementation of adequate control/eradication programs for animal brucellosis, making possible the harmonization of animal health systems worldwide [58]. For animal brucellosis, the OIE is the main standard-setting body publishing.

Factors including cost, time between sampling and achievement of test results, resource requirements, and ease of performance are other aspects that need to be considered in the choice of the best serological methods for the diagnosis of animal brucellosis in a given setting [50]. Indirect diagnostic tests based of humoral immune response against Brucella spp.

Rose bengal test (RBT)

RBT is a slide agglutination technique based on the use of cells of B. abortus S99 or S1119-3 stained with 1% rose bengal as the antigen [59]. It mainly detects IgM and IgG1 [30] mostly produced against LPS from smooth Brucella [60]. The antigen is buffered at an acidic pH (3.65 ± 0.5) in order to limit the agglutination due to IgM increasing the specificity of the technique [53]. The main advantage of this test is its high sensitivity, which makes it very suitable as a first screening test for determining the presence/absence of infection at the herd level [59, 61, 62]. The sensitivity (Se) and specificity (Sp) of this test have been extensively evaluated in different epidemiological scenarios in cattle (with Se estimates ranging from 53% to 100% and Sp from 79% to 100% [63-70]), small ruminants [Se ranges between 75.8% and 100% and Sp between 68.4% and 100% [66, 67, 71-73]), and pigs (Se estimates from 66% to 100% and Sp from 45.5% to 100% [32, 55, 74-76]). Its few technical requirements, speed for obtaining results, and low cost coupled with its performance have made this diagnostic tool one of the most widely used tests for the diagnosis of animal brucellosis. Its main disadvantages are related to its limited specificity in certain conditions, often due to cross-reacting antibodies derived from previous vaccination or exposure to other Gram-negative bacteria [77]. In many countries, the RBT is used as a first screening tool so that positive reactions are subsequently confirmed with an additional technique applied in series if no evidences of infection exist in the epidemiological unit. Due to its reliability, the RBT is one of the techniques described for international trade in cattle, small ruminants, and swine [59, 62].

Complement Fixation Test (CFT)

This test is based on the ability of the complement (obtained from guinea pig serum) to lyse erythrocytes (traditionally sheep erythrocytes sensitized with hemolysin) in the absence of an antibody-antigen complex. When Brucella-specific antibodies are present in the serum being analyzed, they bind to the Brucella antigens (whole cells) provided externally, forming antigen-antibody complexes that then bind to the complement [78]. In this case, the amount of complement in the reaction decreases, preventing its attachment to the hemolysin and the subsequent lysis of the erythrocytes that are added in the final stage. Procedures for performing this technique are described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [59, 61, 62]. Many studies have evaluated the sensitivity and specificity of this technique, showing a slightly to moderately better overall performance in general compared with the RBT (Se estimates vary from 80.6% to 98.79% in small ruminants [66, 67, 79] and from 53% to 100% in cattle [63, 68, 69], and Sp values range between 65.5% and 100% in small ruminants [66, 67, 79] and between 80% and 100% in cattle [63, 68, 69]). Due to its high performance, the CFT has been broadly used as a confirmatory technique in the programs of eradication and control of brucellosis in cattle and small ruminants (for confirmation of RBT-positive results) and is prescribed for international trade. However, under field conditions, the CFT may show a lower sensitivity than RBT, which makes it a very reliable test at the herd level but more limited at the individual level [35, 80]. The sensitivity of this test is also directly related to the stage of infection of the animal being tested, with higher values for animals in an acute phase than in chronically infected ruminants [81]. The disadvantages of this technique are derived from the subjectivity of its interpretation (especially for low titers), the complexity of its performance, and the unavoidable variability of reagents, procedures, etc., that makes comparison of results difficult [82]. Moreover, false positives may also occur in animals sensitized with Y. enterocolitica O:9 [83] and in those immunized with smooth vaccines (Rev. 1 in small ruminants or S19 in cattle [59, 80]) as in the case of RBT. The natural anticomplementary activity of sheep serum must also be considered because it can lead to the occurrence of false-positive reactions [23]. Furthermore, the swine complement interacts with the guinea pig complement used in the test, resulting in a procomplementary activity that may reduce its sensitivity in pigs [32, 84-86]. Nevertheless, the CFT is still contemplated by the OIE as a diagnostic technique of election for swine [61], with a wide range of specificity estimates (from 40% to 100% depending on the study [32, 87, 88]).

Enzyme-Linked Immunosorbent Assay (ELISA)

Since the first ELISA-based technique in the diagnosis of brucellosis [89], many studies have assessed its usefulness. ELISAs based on the use of LPS as the main antigen are considered the most useful and are widely employed. This has led to the commercialization of many ELISA kits (including indirect and competition ELISAs) for its use in domestic species. Based on currently available information, the sensitivity of ELISA-based techniques is similar or greater than that of RB and FC tests, although the actual values may vary depending on the study (from 67% to 100% in small ruminants [71, 72, 90, 91], from 67.9% to 100% in cattle [63, 65, 68, 69], and from 68.5% to 100% in pigs [32, 55, 74, 75]). The specificity of these tests is also considered very high (from 94.5% to 100% in small ruminants [71, 73, 91, 92], from 90.5% to 100% in cattle [63, 65, 68, 69], and from 76% to 100% in pigs [32, 55, 74, 75]). For these reasons, ELISAs are considered by the OIE adequate tests for the diagnosis of bovine, small ruminants, and porcine brucellosis [59, 61, 62], with the advantages of the possible automation of the technique, its reproducibility, and its objective interpretation (since a quantitative value is obtained as the final result). However, ELISAs based on LPS may suffer from the same diagnostic interference problems due to vaccine antibodies [53] and false-positive serological reactors (FPRS) associated with the presence of antibodies against Y. enterocolitica O:9 or other Gram-negative bacteria (mainly in swine [55] and cattle [70]) as those described for RBT and FCT. Due to these diagnostic limitations, the use of other antigens (e.g., proteins of outer or cytoplasmic membrane) has been evaluated for the development of new ELISA techniques for brucellosis diagnosis. However, in general, the serological response induced by these non-LPS antigens is considered to be heterogeneous and delayed compared with that observed using LPS antigen, which may affect the performance of diagnostic tests in the context of a control and eradication program. Still, extensive research has been carried out in this regard, with numerous studies focusing on the development of ELISAs based on proteins that could help to differentiate vaccinated from infected cattle, sheep, and goats (see section Development of Diagnostic Techniques for the Differentiation of Infected and Vaccinated Animals (DIVA)). Other studies have focused on protein targets that may allow the differentiation of Brucella-infected animals from those exposed to other bacteria, usually Y. enterocolitica O:9, mainly in pigs and cattle (BP26 protein, cytoplasmic protein extracts). However, these tests have generally shown a lower sensitivity than the ELISAs based on the LPS [70, 76].

Other indirect diagnostic tests based on the humoral immune response against Brucella spp.

Besides the abovementioned serological techniques, other tests have been developed for the diagnosis of Brucella spp. in animals. Among those, one of the most frequently used techniques is the fluorescence polarization assay (FPA), considered by the OIE as a valid technique for international trade in cattle, small ruminants, and pigs [59, 61, 62]. This test has similar or superior diagnostic performance compared with conventional techniques (CFT/RBT), being technically easier to perform [50, 75, 79, 93, 94]. Thus, in bovine, its use is recommended instead the CFT [59]. Another technique that has been widely used in certain countries is the standard agglutination test (SAT). This assay shows, however, a lower sensitivity and specificity than others, such as RBT and FCT in small ruminants [46, 95-97]. Still, its usefulness in these animal species has been demonstrated [98]. In cattle, SAT has been widely employed in brucellosis eradication programs as a confirmatory test [63, 99]. However, the OIE does not recommend nowadays the use of SAT for international trade in cattle [59] due to its lower specificity compared to CFT [59]. Nevertheless, SAT (and also the FPA) has demonstrated a higher sensitivity compared with the CFT for the detection of recently infected animals [100]. In swine, SAT has also been widely used [101, 102], and it is currently referred as a confirmatory technique in the EU international trade in pigs, although the OIE highlights that it may show a limited specificity due to the presence of nonspecific IgM antibodies in swine serum [61]. Finally, the milk ring test (MRT) has demonstrated its usefulness to detect specific antibodies against Brucella spp. in bovine milk samples used at herd level [59, 103], although its sensitivity may be impaired when it is used in large herds, and its use in small flocks is not recommended due to the expected low specificity [59, 62]. Indirect diagnostic tests based on the cellular immune response against Brucella spp.

Brucellin Skin Test (BST)

This diagnostic test involves the intradermal injection of a mixture of cytosolic proteins generally extracted from a B. melitensis rough strain (e.g., B. melitensis B115-Brucellin INRA) [104] that will induce a delayed hypersensitivity reaction (Type IV) [35] if the animal has previously contacted the bacteria. Recent studies have studied the usefulness of B. abortus ΔmanBcore as an alternative source of antigens for the BST for swine brucellosis diagnosis [87, 105]. The inoculation is usually performed in the lower eyelid in small ruminants [62], in the caudal fold, in the skin of the flank or the side of the neck in cattle [59], and in the base of the ear or the side of the tail in pigs [61]. Free-LPS antigens are required to prevent the subsequent induction of cross-reacting antibodies that may interfere with the traditional serological tests [81]. The main characteristic of BST is its high specificity, which makes it a useful tool to elucidate problems due to FPSR caused by Y. enterocolitica O:9 [106, 107], especially in brucellosis-free areas [25, 44], and as a complementary technique to serological tests [108]. However, occasional false-positive reactions due to Ochrobactrum anthropi infection have also been described [109]. In addition, its use is exclusively indicated in unvaccinated animals. This technique may be of particular interest to detect animals in the early and chronic stages of infection [23, 108], but its limited sensitivity makes its interpretation at the individual level difficult. Still, it is considered a suitable tool for Brucella diagnosis at herd level. Herd history, clinical signs, and serological or bacteriological results should be considered in the interpretation of the BST results. In ruminants, BST is recognized as an alternative technique for international trade [62]. In the case of swine brucellosis, it is not currently regarded as an official test, although its usefulness is supported by the EFSA and the OIE as a complementary diagnostic test [32, 61].

Interferon Gamma (IFN-γ) Detection

The interferon gamma is one of the most important cytokines involved in the cellular immune response against Brucella [110-112]. The usefulness of the in vitro quantification of IFN-γ produced by cells from naturally infected animals for monitoring the cellular immune response against this pathogen has been studied in cattle [113], porcine [107], and sheep [114, 115]. The levels of IFN-γ measured in samples stimulated with a Brucella-specific antigen (and that should be especially high in cases of previous contact with Brucella spp.) are typically quantified using an ELISA. This technique has been suggested as a complementary test to the serological techniques routinely used for the diagnosis of brucellosis in ruminants in the case of false-positive reactors due to its specificity [113], although there is still some controversy about its usefulness as a routine diagnostic technique for brucellosis.

2.2.2. Direct diagnostic tests

These techniques are based on the direct detection of the etiologic agent or its genetic material in clinical samples from infected animals. Brucella isolation is the gold standard for confirmation of infection [59, 61, 62, 116], and its use is recommended to improve the efficiency of eradication plans [25]. The use of specific staining methods (e.g., Stamp’s method) in clinical specimens may reveal the existence of Brucella in clinical samples, although the limited sensitivity and specificity (especially due to the potential presence of other abortifacient pathogens such as Chlamydia abortus and Coxiella burnetii [62] that may lead to false-positive results) of this diagnostic approach can compromise its usefulness for the routine laboratory diagnosis of brucellosis. Bacteriology

The World Health Organization classifies the Brucella genus bacteria as a risk agent III [62], and therefore high laboratory and training requirements are needed to handle the pathogen and potentially contaminated samples. The preferred samples for the direct detection of Brucella spp. are as follows: (i) in live animals: vaginal swab, milk, placenta, and fetus aborted samples (especially lung, spleen, and stomach contents) and (ii) postmortem: the reticuloendothelial system, udder, uterus, testis, and epididymis [25, 59, 61, 62, 117]. The possible contamination of clinical samples requires the use of selective media, frequently Thayer-Martin and Farrel media, for the isolation of Brucella spp. [59, 61, 62]. However, the presence of some components in the Farrel medium may have an inhibitory effect on some B. abortus and B. melitensis strains as well as in the B. suis biovar 2 [118]. Thus, the combination of more than one selective culture media and inoculation of at least two plates of each sample, including also a nonselective media for the isolation of B. suis biovar 2, may help to increase the chance of isolation of the pathogen [119]. The addition of serum or blood (usually from horse) to the media promotes the growth of Brucella spp. [82] and is necessary in the case of B. abortus biovar 2 [59]. Moreover, some strains of B. abortus (biovars 1-4 and 9) need a 5-10% CO2 atmosphere [59]. Microbiological culture has a limited sensitivity in certain epidemiological situations, such as chronically infected animals that have typically low bacterial load in their samples. For this reason, negative culture result should not be considered as definitive evidence to rule out Brucella infection in an animal/herd [29, 120]. In fact, the sensitivity of culture is highly variable, depending on the stage of infection, the specimens analyzed, and the number of samples cultured [100, 102, 118, 121-123]. Molecular detection

DNA-based techniques allow the detection of nonviable Brucella or highly contaminated samples that may be challenging if handled using a traditionally culture-based diagnostic approach. In addition, molecular techniques may be applied at large scale through the use of automated equipment. PCR techniques are also routinely used for identification of Brucella-compatible isolates cultured from clinical samples. A considerable number of molecular techniques based on the PCR amplification of Brucella spp. DNA has been described, although often these techniques have not been fully validated on field samples as direct detection tests, hampering their implementation as routine techniques for Brucella detection [124]. Despite the high homology of DNA among Brucella species, different PCR protocols to identify different species/biovar and even vaccine/field strains have been described [125-133]. The efficiency in the DNA extraction procedure (which depends on the nature of the sample [134]) determines substantially the results of the PCR assays [135-137].

2.3. Immunization

Since Eduard Jenner described the first vaccine against smallpox in 1796, the immunization against infectious agents has become a cornerstone in the control of many of the most important infectious diseases. As general rule, vaccination efforts are often focused in the most susceptible individuals in order to stimulate a protective immune response against the pathogen of interest. However, in the case of animal brucellosis, vaccination campaigns target a less susceptible population (nulliparous nonpregnant young sheep, goats, and cows), while vaccination of the most susceptible subset is avoided due to the side effects of vaccination of pregnant animals (abortion, bacteria excretion, environmental contamination, etc.) [26].

The characteristics of the “ideal vaccine against brucellosis” include the following: (i) to induce a solid and long-lasting protection against the infection by different Brucella species without the need of re-vaccinations, (ii) to be innocuous regardless of the reproductive stage of the animal (so that there is no induction of abortion in pregnant animals, and mass vaccination can be applied if needed), (iii) to have no or very residual virulence for human and be susceptible to the antibiotics typically used to treat human brucellosis, (iv) to avoid the induction of cross-reacting antibodies in the serological techniques traditionally used in control/eradication programs, (v) to be affordable, and (vi) to possess stability at different environmental temperatures [138, 139]. Unfortunately, this ideal vaccine is far from those currently available for the control of animal brucellosis. For example, in the case of small ruminant brucellosis, Rev. 1 has been recognized as the most effective vaccine currently available considering its efficacy to prevent the abortion and transmission of B. melitensis, but it cannot be applied in pregnant females and is pathogenic for humans, among other nondesirable side effects [139]. The smooth S19 strain is the most widely employed vaccine in the case of bovine brucellosis, and even though it is currently considered the reference strain, it presents similar limitations as the Rev. 1 despite its demonstrated efficacy. For these reasons, the rough strain RB51 is increasingly used in some regions of the world as an alternative for vaccination against bovine brucellosis since its use does not induce the production of cross-reacting antibodies, even though its efficacy is still under discussion in certain epidemiological situations [140]. In general, the inability to vaccinate pregnant animals is a major disadvantage in animal brucellosis vaccination since it complicates achieving a quick increase in the proportion of the resistant subset of the population, which could lead to an average benefit-cost ratio of 3.2 (2.27-4.37) by reducing (52%) the transmission between animals by means of mass vaccination [21].

The success of the use of live vaccines for immunization against animal brucellosis is based on a balance between an adequate colonization of the host, triggering a solid protection against infection with other Brucella field strains, and a limited replication that minimizes the residual virulence of these vaccine strains [141]. Although immunological mechanisms induced by living vaccine have not been completely elucidated, live vaccines should stimulate the innate immunity, activate CD8+ and CD4+ cells, and generate an adequate population of memory cells, among other mechanisms, to induce a solid protection [139].

Most of the drawbacks associated with the use of live vaccines could be overcome with the use of killed bacteria or subunit vaccines; however, the ability of these inactivated vaccines to provide a solid and long-lasting immune response against Brucella has been traditionally considered lower than that triggered by live attenuated vaccines. New approaches are being investigated to elude the main drawbacks of inactivated vaccines to induce protective immunity in domestic livestock.

In the following section, the main drawbacks of currently available vaccines (Rev. 1 for SRB and S19/RB51 for BB) are described, as well as some of the new approaches followed to solve some of these shortcomings.

2.3.1. Residual virulence of attenuated live vaccines

The pathogenicity of Rev. 1 and S19, evident in its ability to induce abortion, has been a limitation since they were first used. However, in the case of Rev. 1, some authors have reported significant differences in its residual virulence. In a study performed to assess the immunogenicity and residual virulence of Rev. 1 strains recovered from different geographic origins, important differences between strains were identified [142], which may explain, at least in part, the diversity of results sometimes reported using this strain. For this reason, according to the OIE recommendations, strict quality controls must be maintained during the Rev. 1 production process in order to confirm that all batches have the typical characteristic of the original B. melitensis biovar 1 Rev. 1 strain. Residual virulence of Rev. 1 and S19 is reflected in the following sections. Abortifacient effect

The ability of Rev. 1 to induce abortions was reported in the first studies, around the middle of the last century [143]. This adverse effect is especially significant when females are immunized around midpregnancy [144]. The abortifacient effect of the S19 strain has been demonstrated, although it is considered reduced and lower than that observed after Rev. 1 vaccination in general [140]. The appearance of a small percentage of vaccinated animals that may remain persistently infected with the S19 strain has also been described and may lead to abortions in adulthood [145]. In addition, vaccination may involve the excretion of the vaccine strain in milk and vaginal secretions [146, 147]. Even so, although the possible excretion of Rev. 1 during the lactation has been demonstrated, there is some disagreement about the relevance of this phenomenon [148].

Different strategies have been explored in order to avoid the abortifacient effect of Rev. 1 and S19, as described in the following sections.

Restriction of vaccination to replacement females (nulliparous, nonpregnant)

The protection induced by vaccination at full doses in 3-6-month-old animals using Rev. 1 [149, 150] and in 3-8-month-old animals using S19 [59, 151] is sufficient to induce a long-lasting protection. However, this control strategy may be problematic in certain epidemiological situations (e.g., high prevalence of brucellosis when mass vaccination is the only strategy to control the disease [24]).

Application of reduced doses of vaccine

Different studies have demonstrated the suitability of the immunization with reduced doses as an alternative to full dosages of vaccines, assuming that the afforded immunity was adequate and/or the abortion rate associated with the vaccination was significantly lower [59, 152-154]. However, field results obtained after the application of Rev. 1/S19 at reduced doses has led to a divergence of opinions among different experts about its usefulness and implications: many authors have reported a significant number of reproductive failures/vaccine excretion [155-157], advising against the use of this strategy in brucellosis control programs. In addition, some authors have showed that this immunization strategy using Rev. 1/S19 induces an inadequate protection against B. melitensis/B. abortus infection [156-159]. Additionally, reduced doses may not avoid the induction of persistent antibody titters when Rev. 1 and S19 are applied in adult animals, leading to a diagnostic interference problem with traditional serological techniques [59, 153, 156]. However, other experts have argued in favor of the efficacy of this approach as part of some brucellosis control programs [160-163]. In the case of S19, some experiments have shown that the best protection was obtained by subcutaneous vaccination of calves at full doses followed by conjunctival administration of a booster reduced dose [164].

Modification of route of vaccination

Although the application of Rev. 1 by the conjunctival route has been demonstrated to reduce the number of reproductive failures induced after subcutaneously immunization of pregnant females, the safety of this immunization strategy is not enough to be used regardless of the physiological stage of females [144, 146]. In bovine, S19 conjunctival vaccination does not avoid completely abortions in pregnant females [155], although it is considered that the conjunctival administration in reduced doses (5 × 109 microorganisms) is an alternative route in adult vaccination due to the reduction of abortion rates [59]. Other side effects

In addition to the induction of reproductive failures, other adverse effects, as transient periods of fever and anorexia or swelling at the vaccination site, have been reported after Rev. 1 vaccination [31]. Some studies have showed a risk of environmental contamination with the vaccine strain after Rev. 1 vaccination of young females, suggesting these animals are shedding the Rev. 1 strain and could therefore be a source of infection for other susceptible individuals [165, 166]. However, other authors have reported that conjunctival vaccination of nonpregnant animals immunized is safe for the environment [167, 168]. Some occasional contradictory effects (as orchitis) in billy goat and ram have also been reported [169, 170]. Regarding to the S19 vaccine, other side effects reported include the appearance of arthropathies associated with type III hypersensitivity reactions [171], persistent orchitis in males [153], and significant reduction in milk production and udder infections [140, 172].

2.3.2. Zoonotic potential and antibiotic resistance of attenuated living vaccines

A limited number of human brucellosis due to Rev. 1 [173, 174] and S19 [175-177] infection has been reported. The risk of infection for human and environmental contamination is nevertheless reduced if the adequate biosafety practices during handling these live vaccines are followed. Rev. 1 and RB51 carry antibiotic resistance genes to streptomycin and rifampicin, respectively, which are used in the treatment of human brucellosis. In the case of S19, resistance to penicillin G (associated with an increased virulence in mice) has been evidenced [178].

2.3.3. Instability of vaccine strains

The possible instability of Rev. 1 is due to its tendency to dissociate into a rough phenotype, reducing its effectiveness in the field [138]. Several strategies such as the addition of 5% of serum (from horse, cattle, or rabbit) on the solid medium used for Rev. 1 production have been demonstrated to be useful to prevent this dissociation [179]. Biological quality and efficacy of S19 may be affected by inadequate subculture or maintenance conditions [180]. In the field, the main strategy to prevent this instability is the strict control of refrigeration temperatures for storage during all the process until the moment of inoculation (what could be problematic in certain circumstances, leading to the application of low-quality vaccines) [181]. In addition, preliminary observations also suggest that a reversion to a more pathogenic phenotype of the Rev. 1 strain is also possible [166], although the genetic stability and homogeneity of Rev. 1 strains is considered demonstrated [182].

2.3.4. Diagnostic interference in serological techniques

The smooth LPS antibodies induced after Rev. 1 and S19 vaccination are indistinguishable from those triggered after infection with a smooth field Brucella strain. The Rev. 1-derived antibodies may be detected even 4 years after vaccination depending on the age of the animal at vaccination, the immunization doses, and the serological technique being used [183]. For this reason, vaccination using smooth Rev. 1 and S19 creates a diagnostic interference problem when test-and-slaughter (T&S) programs based on the use of traditional serological techniques are in place, complicating the combination of these two strategies. The following sections present three possible approaches to overcome this diagnostic interference problem. Reduction of the serological response induced by Rev. 1 vaccination

  • Application of reduced doses of vaccine (see section Application of Reduced Doses of Vaccine)

  • Restriction of vaccination to replacement females [nulliparous, nonpregnant; see section ´Restriction of Vaccination to Replacement Females (Nulliparous, Nonpregnant)].

  • Modification of the route of vaccination

The subcutaneous route, traditionally used for the immunization with Rev. 1 and S19 in small ruminants and cattle, respectively, triggers a solid and long-lasting serological response (at least up to 20 months after vaccination in goats [184] and 22 months in cattle [185]). However, in the last 30 years, the usefulness of the alternative use of the conjunctival route has been demonstrated in cattle [164, 186] and small ruminants [187]. Vaccination of young animals using Rev. 1 inoculated by the conjunctival route induces a limited serological response but is able to trigger an adequate protection for at least the two first pregnancies following vaccination) [188]. The serological response induced by conjunctival Rev. 1 vaccination is more long-lasting in adults, therefore leading to the potential occurrence of diagnostic interference problems [187], although these would be still more limited than those observed after its application by the subcutaneous route [189].

In the case of the S19 vaccination, the OIE suggests an alternative immunization protocol based on the application of 5 × 109 CFUs conjunctively in bovines regardless of the age of the animal [59]. Thus, as mentioned above, the vaccination protocol based on subcutaneous application of S19 during calfhood and conjunctival revaccination using reduced doses in adult animals has been proved as a suitable strategy for the control of BB in endemic populations, which avoids the need for slaughter of false-positive reactors [164] Development of diagnostic techniques for the Differentiation of Infected and Vaccinated Animals (DIVA)

Different studies have been carried out to identify immunogenic non-LPS components that could allow the development of diagnostic techniques able to detect the serological response induced by Brucella field strains only. Some studies have showed promising results, but most of the authors also report limitations (such as less intense and more heterogeneous antibody response, lack of information about diagnostic performance under field conditions, etc.) that make their routine implementation on animal brucellosis control/eradication campaigns difficult. In the case of S19, the use of competition ELISAs coated with S-LPS allows the removal of most false-positive responses due to vaccination-derived cross-reacting antibodies [59, 93, 190, 191].

The use of several non-LPS Brucella components as the main antigen for DIVA serological techniques has been described, including the following:

  • Proteins. The detection of immunogenic proteins involved in the humoral immune response of the host in the outer membrane of Brucella was the first step in the development of alternative serological methods for brucellosis diagnosis. In the case of B. melitensis, CP28 (also called BP26) was recognized as a good candidate to distinguish between Rev. 1 vaccinated and infected animals [192]. Several indirect [193, 194] and competitive [195] ELISAs have been developed to assess the suitability of CP28 as a diagnostic target for small ruminant brucellosis diagnosis, showing a lower diagnostic performance than that detected using ELISAs based on the LPS antibodies response [196]. In the case of B. abortus, different protein antigens (N-formylperosamine O-polysaccharide-protein conjugate [197], an extract of cytoplasmic proteins of Brucella and an 18-kDa cytoplasmic protein [198]) have been described as suitable candidates for the development of new immunological tests for screening and infection confirmatory diagnosis. Additionally, Pajuaba et al. showed that the AHRPO protein (protein A-horseradish peroxidase) is an adequate conjugate for the development of an indirect ELISA (that uses B. abortus S-LPS as an antigen) to differentiate S19 vaccinated and B. abortus-infected animals due to a preferential detection of the IgG2 isotype, a valuable marker of Brucella infection [199].

  • Polysaccharides. The native hapten, one of the polysaccharides present in the surface of smooth Brucella strains, has also been used as a potential antigen in an agar gel immunodiffusion (AGID) test to solve the potential diagnostic interference problem. When antibodies against this polysaccharide are present (samples from B. abortus/B. melitensis-infected ruminants), a ring of precipitations appears in the agar [200, 201]. If samples from vaccinated (and uninfected) animals are analyzed several months after vaccination, no antibodies against the polysaccharide would be detected. In adult cattle, subcutaneous vaccination with reduced doses does not produce positive reactions (except for those animals that are infected and excrete the bacteria in milk [202]). The AGID test has demonstrated its usefulness to solve diagnostic interference problems in bovine and small ruminants [77, 203, 204], although some authors have also reported a sensitivity too limited for its wide-scale application [53]. This technique has also been demonstrated to be suitable to elucidate cases of FPSR due to Y. enterocolitica infection in cattle [70]. Use of rough vaccine strains (lack of O-PS or O-PS defective strains)

Rough Brucella strains are naturally devoid of the O-PS, which confers them a more granular and dull surface compared with those carrying complete S-LPS (smooth strains). Due to the lack of antigenic O-PS, these rough mutants may not induce anti-O-PS antibodies and, therefore, do not cause diagnostic interferences in most serodiagnostic tests. The rough phenotype can be observed by crystal violet staining (rough strains uptake the staining turning to red/violet) or autoagglutination in acriflavine solution [140]. In 1997, a WHO Consultation on the Development of New/Improved Brucellosis Vaccines encouraged the need of studies on live attenuated rough vaccines as an alternative to smooth strains for immunization against brucellosis [205]. Mutant rough Brucella strains are obtained by (i) natural dissociation of smooth (S) to rough (R) phenotype and subsequent repeated in vivo or in vitro passages of R mutants or (ii) genetic modification of the sequence of genes involved in the synthesis/transportation of component(s) of the smooth LPS [140]. Despite the potential advantages associated with the use of rough strains as vaccine candidates, some potential undesirable traits may make their application in the field difficult:

  • The attenuation of R mutants has been associated to modifications in the outer membrane of Brucella and, therefore, possible changes in their interaction with components of the immune system of the host [206]. If the attenuation is too high, the rapid clearance of the R mutants in the host may lead to an insufficient protection [140]. However, natural rough Brucella species (B. canis and B. ovis) are virulent for their preferred hosts, and in fact, the role of LPS in the pathogenesis of Brucella is controversial: although the protection against Brucella is mainly mediated by the cellular immune response (triggered primarily by bacterial antigenic proteins), the humoral response (antibodies) may also have a role in the resistance against Brucella, as demonstrated by passive immunity experiment with sera against the LPS [207-209] and even with antibodies against the rough B. melitensis B115 strain [210] (see section Control of Small Ruminant Brucellosis).

  • The diagnostic interference associated to the smooth phenotype of Brucella may not be totally avoided with the use of rough strains since animals vaccinated with R strains have showed occasional reactions to the S-LPS ELISA [211].

  • The possible zoonotic potential of rough Brucella strains may limit their large-scale use. In the case of human brucellosis due to rough Brucella strains, traditional serological techniques, based on the detection of antibodies against S-LPS, may not detect the infection leading to misdiagnosis [140]. A possible alternative in this case would be the use of specific techniques for the detection of antibody response against rough Brucella, such as the CFT developed for the detection of rough B115 B. melitensis infection in sheep [212].

In the case of bovine brucellosis, the suitability of several rough vaccines (45/20, pgm mutant, RB51, Brucella abortus strain 82) for the control of the infection due to B. abortus has been evaluated [213-216]. However, RB51 vaccine could be considered the main representative example of the potential usefulness of rough Brucella vaccines in the battle against animal brucellosis. RB51 (R: rough; B: Brucella; 51: identification number of the laboratory of origin) is a stable rough rifampicin resistant B. abortus strain produced after repeated passages of B. abortus strain 2308 on trypticase soy supplemented with 1.5% agar and varying concentrations of rifampin or penicillin [215]. No O-PS antibodies were detected in rabbits, goats, and cattle after immunization with this strain [215, 217, 218], although some authors have reported low level of M-like O-chain in vaccinated species [219] and certain apparent anamnestic responses have been reported [220]. The induction of none to very limited vaccine-mediated abortions has been reported in cattle [221, 222], although contradictory evidences have also been reported [223]. In cattle, RB51 triggered an adequate protection against infection with virulent B. abortus strains [151, 224, 225], but certain concerns have been raised regarding its suitability in certain epidemiological situations in the field (see section Control of Bovine Brucellosis). RB51 has also been evaluated as a tool for the control of small ruminant and swine brucellosis. In the case of B. suis, some authors have demonstrated its usefulness preventing abortion in swine [226], although recent studies have demonstrated the inefficacy of parental RB51 vaccination to induce humoral or cell-mediated immune responses or to protect against abortion in a virulent challenge with B suis in domestic pigs [85]. Protection induced by RB51 vaccination against B. melitensis was also insufficient to consider it as a potential candidate in the prevention of ovine brucellosis [227], although the results regarding the suitability of this vaccine strain for the immunization of goats are controversial [140, 228, 229].

Many attempts have been conducted to achieve a stable immunogenic rough vaccine against B. melitensis using genetic engineering [VTRM1- rfbU mutant, rpoB mutant, wa**, and wzm mutant, B115) [206, 211, 230-232]. In the case of swine brucellosis, a recent study of Stoffreger et al. has demonstrated that a rough B. suis strain (353-1), isolated from urine of a feral boar and prepared by propagation from the original isolation on Tryptose agar containing 5% bovine serum (TSA) at 37°C and 5% CO2, can induce a significant immune response and confer a partial level of protection from a challenge with a virulent B. suis [233]. However, no rough vaccine candidate is currently recognized as a suitable alternative for immunization of sheep/goats and swine in the framework of an animal brucellosis control program.


3. Control of bovine brucellosis

The OIE has established the following requirements for a country/zone to be considered as free from this disease: (i) BB is declared notifiable; (ii) an official veterinary control is established in the entire bovine population, in which flock prevalence of disease is lower than 0.2%; (iii) no vaccination has been performed in the last 3 years (at least); (iv) all herds are subjected to periodical serological testing; (v) all reactors are culled; and (vi) new animals introduced in the region belong to officially brucellosis-free (OBF) herds (or free of brucellosis with vaccination). Some countries (France, Germany, Norway, Sweden, The Netherlands, Japan, Canada, Australia, and New Zealand, among others [59]) are considered to be OBF, but the disease is still present in many others despite the implementation of control/eradication programs. In general, measures against BB are based on vaccination (when the prevalence is high, in the initial steps of the control strategies) and test-and-slaughter programs (or herd depopulation when the disease prevalence is already very low and economic and technical resources are available) in the final stages of the eradication process prior to the achievement of the OBF status. These measures (vaccination, test and slaughter programs, and herd depopulation) alone cannot however be fully effective in eradicating the disease without the additional implementation of other complementary prevention measures, such as control of animal movements, use of surveillance systems, adequate laboratory support, etc.

Even though the role of wildlife reservoirs in the epidemiology of bovine brucellosis is considered minimal in most countries in the world, several wild species in certain scenarios can act as reservoirs of the disease for cattle, such as wild bison elk in the Greater Yellowstone Area (USA) [234]. In these situations, wild populations should be considered in the design of BB control programs.

In the case of BB, two vaccines are available for the control of B. abortus in cattle: the S19 (smooth) and the RB51 (rough) strains, which have demonstrated their effectiveness in the reduction of the number of abortion, transmission, excretion, etc., in certain epidemiological situations [235].

The S19 strain has been the main vaccine used against bovine brucellosis in many countries for more than 50 years. Numerous studies have demonstrated the usefulness of S19 calfhood vaccination with full doses (1010 CFUs) to protect them against B. abortus infection during their whole productive lifespan [236]. However, as mentioned before, its smooth nature may lead to the induction of O-PS antibodies that may persist until the adulthood, causing a diagnostic interference problem. Nevertheless, adult vaccination may be occasionally recommended in certain scenarios (high prevalence settings in which a rapid impact on disease spread is needed, large herds in which test-and-slaughter strategies are not feasible). Still, adult vaccination remains an emergency measure since the induction of vaccine antibodies and the possible abortifacient effect of S19 in pregnant cows are two very important side effects that would be associated with it. The use of reduced doses of S19 vaccine (109 CFUs) in adults can partially limit those side effects, but its full usefulness is debatable [214, 237]. Immunization with RB51 vaccine (rough strain with a minimal expression of O-PS) for the control of B. abortus infection has emerged as an alternative to S19/stamping out in certain scenarios [238]. However, its true usefulness and its ability to induce a degree of protection equivalent to that induced with the S19 vaccine are still under discussion. The safety and protection afforded by RB51 against infection with B. abortus have been demonstrated in experimental conditions [224, 239, 240], but some of these results are still considered controversial [140]. Field evidences suggest RB51 could be considered a useful complementary tool for BB control [151, 238, 241], but the impossibility of comparing the progress achieved by strategies with and without RB51 vaccination under the exact same conditions in most cases impairs the evaluation of the relative contribution of RB51 vaccination to the overall success of an eradication program [242]. Recent studies have showed a potential beneficial effect of an RB51 booster vaccination in adult cattle after S19 calfhood vaccination to control B. abortus infection [243].

Even though B. abortus is undoubtedly the main concern when dealing with infection in cattle, B. melitensis is also a potential etiologic agent of brucellosis in bovine. The control of B. melitensis infection in cattle is hampered by the lack of information on important aspects of its epidemiology [244]. Outbreaks due to B. melitensis in cattle are often attributed to the presence of infected small ruminants in the surrounding area [245], suggesting that the key for the control of this pathogen in bovine will be the control of the disease in ovine and caprine flocks, as for human brucellosis.


4. Control of small ruminant brucellosis

Even though the most important factors that have to be considered for the control of SRB have been well characterized, socioeconomic factors have influenced the choice of the most suitable control measures in most of the endemic areas. For example, the existence of pastoral ecosystems, a traditional management practice in low-income areas where B. melitensis is endemic, has contributed to the perpetuation of the disease due to the difficulty of detecting the disease in the early stages of infection and of implementing control measures [246]. In addition, management practices favoring the mixing of animals with different origins (for example, the existence of communal grazing pastures) modify the traditional concept of minimal epidemiological unit of intervention (an essential key to consider by decision makers of animal brucellosis control programs). The minimal epidemiological unit is defined as “any number of animals that are held, kept or handled in such a manner that they share the same risk of exposure to brucellosis” [247] and can therefore include the flock or supraflock levels.

Although extremely important, the prevalence of brucellosis is not the only issue to consider for decision makers: the organization of the veterinary services, the availability of a suitable animal identification system and of the economic and technical resources that these measures require, the involvement of producers, veterinarians, and administrative authorities, etc., are all key aspects that must be evaluated [24]. When the prevalence of brucellosis is high and/or the socioeconomic resources are limited, the vaccination is the most suitable tool for the control of the disease. Despite its drawbacks, Rev. 1 is the best currently available vaccine to immunize sheep and goats against B. melitensis. In order to minimize the diagnostic interference problem due to Rev. 1 cross-reacting antibodies, a restricted vaccination strategy has been implemented in most of the regions where test and slaughter policy is applied as part of the SRB control program. The vaccine is administered by the conjunctival route in young (<6 months) female animals at doses of 0.5-2 × 109 CFU/animal (also controlling the impact of the abortifacient effect of Rev. 1). Some authors [24, 31] have suggested that restricted vaccination may be insufficient in certain epidemiological situations (such as high prevalence regions or where nomadism is practiced) and mass vaccination should be implemented instead. The immunization at full doses by the conjunctival route during the prebreeding period and late lambing season would be the most suitable approach for whole flock vaccination because it may prevent reproductive failures due to Rev. 1 vaccination [156]. Blasco et al. [24] have described two methods to carry the whole flock vaccination in the case of sheep and goats: (i) mass vaccination of males and females every two years avoiding lambing period (and considering 15-25% annual replacement) and (ii) restricted vaccination of replacement animals (at least 8-10 years) except the first year when all animals would be immunized regardless of their age. When the prevalence has decreased, a restricted Rev. 1 strategy could be then implemented. In general, a vaccination program against SRB should last at least 8-10 years in order to assure an adequate vaccine coverage [24]. The correct identification of vaccinated animals is an important factor to achieve this vaccination coverage that will grant an adequate immunity at the flock level [33].

Despite the success achieved thanks to Rev. 1 vaccination in many regions of the world, the drawbacks associated with its use have prompted the study of alternative vaccines against B. melitensis: DNA vaccines, subunits vaccines, outer membrane vesicles, smooth B. melitensis mutants (BP26, P39, Omp25, and ΔpurE201), nondividing but metabolically active gamma-irradiated Brucella melitensis, and attenuated live rough vaccines [230, 231, 248-254]. Among rough vaccines against SRB, the B115 strain has demonstrated to induce a solid protection not only against B. melitensis but also against B. ovis and B. abortus in the murine model. An abortifacient effect was demonstrated in sheep after subcutaneous vaccination with B115, although the interference diagnostic problem was drastically reduced [255]. Still, the possible occurrence of reversions of its phenotype (rough to smooth) in vivo could affect its attenuation, thus highlighting the need of further studies to better determine the stability of B115 in sheep before giving it further consideration as a possible candidate for SRB vaccination.

When certain epidemiological indicators (disease prevalence, number of cases of human brucellosis, etc.) suggest the control of brucellosis has been achieved, the next step is to success in the eradication of the disease [24]. A restricted Rev. 1 immunization strategy should be implemented exclusively when the prevalence is already low after 6-12 years of whole-flock vaccination, a strict control of animal movements and an accurate animal identification system exist, and veterinary services and economic resources are available. The change from mass vaccination to restricted vaccination is a critical step in the control/eradication programs since it may suppose the culling of a high number of false seropositive adult vaccinated animals, even if conjunctival vaccination is performed. Two different approaches have been proposed to minimize this shortcoming [24]: (i) lack of testing for two years after the change from mass to restricted vaccination (after which a strict test and slaughter policy would be implemented, where all CFT-positive animals are culled and their flocks of origin retested until 100% of animals are seronegative at least two consecutive tests) and (ii) the implementation of a serological test capable of differentiate vaccinated from infected small ruminants as part of the test-and-slaughter strategy as soon as the whole flock vaccination is stopped (such as the radial immunodiffusion tests whose usefulness has been discussed before). The restricted vaccination should be maintained after the achievement of a close to zero brucellosis prevalence. After that, if the epidemiological situation is maintained and the risk of reintroduction of B. melitensis has been minimized, the prohibition of vaccination and the implementation of an eradication program based exclusively on test and slaughter policy could be proposed. Vaccination coverage, the implementation of other complementary measures, and the availability of an effective monitoring strategy should be considered by decision makers to establish the moment of vaccination ban [34]. The availability of economic and technical resources (economic compensation for farmers, strict animal movement control, etc.) is a cornerstone to ensure the success of a test-and-slaughter strategy in regions with close to zero disease prevalence.


5. Control of swine brucellosis

Porcine brucellosis is a notifiable OIE-listed disease [256]. However, in many countries, no specific control and eradication programs are implemented, and there is no obligation to conduct monitoring/surveillance strategies on the swine population for Brucella infection. Therefore, the actual prevalence of porcine brucellosis in many areas is not exactly known and probably is often underestimated. For example, in the case of EU Members States, the estimates of the prevalence of swine brucellosis vary depending on the region, but the current situation of B. suis in swine population is not totally recognized due to the lack of systematic epidemiologic data [32]. One of the main differences in the control and eradication of this pathology in swine compared to ruminant brucellosis is the lack of availability of safe and effective vaccines against B. suis as Rev. 1 for B. melitensis and S19/RB51 for B. abortus [32, 61]. Nowadays, the only country where vaccination against swine brucellosis is applied is China, where a national control program against this disease (mainly caused by B. suis biovar 1) was initiated due to the high rate of human brucellosis cases and the economic losses in animal production. In addition to animal vaccination, other strategies such as culling of all aborted females, separation and removal of infected animals, and quarantine policies were also implemented [257]. Since 1981, pigs have been immunized in China with a live attenuated B. suisS2 vaccine. In some brucellosis-infected areas with low prevalence, vaccination was only applied to young livestock so that test-and-slaughter programs could also be implemented. In other areas in which this was not feasible, an intermittent vaccination policy was applied, and the vaccinated animals were not tested to avoid the diagnostic interference problems caused by the use of this vaccine. The S2 vaccine can be administered by parenteral route or per os, although it should be applied at high doses (20 × 1010 FCU/animals) in two doses [257]. One of the side effects of this vaccine is that it can cause abortions in pregnant sows, and despite its wide use in China for decades, OIE does not recommend its use for the control of swine brucellosis, partly due to the absence of trials demonstrating its efficacy and safety in controlled conditions [61].

In general terms, although in most countries there are no specific plans for the control and eradication of porcine brucellosis, the most frequent approach for swine brucellosis control is the test and slaughter strategy, similar to that used in ruminants. Thus, eradication requires the identification of infected animals, the progressive elimination of reactors from the herd, and their replacement with noninfected animals (testing all animals and applying a quarantine period) [32]. The application of whole-herd slaughter when reactors are detected should be considered as one strategy that would reduce the risk of circulation within the drove and its spread to other holdings due to undetected infected animals. However, this measure is often not feasible because of the economic implications for farmers. On the other hand, whole-herd depopulation and repopulation with noninfected animals must be implemented in countries considered free of the disease [258]. An example of a country where this measure is used as part of its control an eradication program is the USA, where B. suis has been eradicated from commercial pigs [259], thanks to the great efforts invested in the last decades. In 1972, the U.S. Department of Agriculture National Brucellosis Eradication Program implemented in ruminants was expanded to cover swine herds, based on the serological testing and removal of reactors. Depending on the epidemiological situation, three strategies were implemented [260]: (i) whole-herd depopulation of infected herds and repopulation, recommended for commercial herds and seed stock producers who wish to eliminate swine brucellosis from their population rapidly, and often the solution in the final efforts at eradication; (ii) exceptionally, in herds with only one or a few reactors (and no clinical signs of swine brucellosis), frequent test and removal of reactors, although this option was not generally recommended; and (iii) offspring segregation: this plan was recommended where valuable bloodlines had to be saved and weaned pigs (usually negative when tested at weaning) were allowed to be moved to separate, clean premises (animals had to be retested at least once prior to breeding to tested, clean boars).

As part of a control program, certain conditions and testing are required to classify a herd as swine brucellosis free. Thus, herds may be validated as swine brucellosis free by conducting a complete herd test with negative results. Validation may be maintained by periodical testing of the whole herd with negative results. Besides USA, some other countries in America (such as Cuba and Panama) [261], where porcine brucellosis is present, have also implemented control and eradication programs based on these same approaches. Some countries and regions offer the farmers the option of implementing voluntary programs covered by the authorities in order to certify the flocks as swine brucellosis-free herds. These voluntary programs are based on the same principles as those used in the eradication and control programs, consisting of periodic serological testing of all animals in the herd and removal of reactors. This measure has been implemented in Argentina, being mandatory for local genetic suppliers and breeding animals destined for sale, fairs, auctions, and exhibitions [262]. Another example of voluntary program is in Australia, accepted by all states and nowadays implemented in Queensland, where B. suis is an enzootic disease of feral pigs [263]. Farms included in the Australian voluntary program should buy breeding stock only from herds registered in this scheme. If infection is detected in the herd, then the accreditation is withdrawn until all reactors are removed and the herd tested back to accreditation standards [264]. Another fundamental key for the control of swine brucellosis is the control of artificial insemination centers because they may be an important source of infection to many animals [265]. Therefore, control measures implemented in these centers are especially restrictive and have specific regulations for each country or region. These measures are based on serological testing and quarantine of all introduced animals (that must come from accredited B. suis-free farms), continuous serological monitoring of the whole population of the center, and preparation of semen doses following certain requirements [32].

Control of wildlife reservoirs is an important part of the swine brucellosis control and eradication in regions where the disease is widespread in wildlife and may get in contact with the commercial swine population. For example, in USA, where swine brucellosis remains endemic in feral swine [266], the control of PB in this wild species is included in the current program of control and eradication, with specific measures regarding the restriction of feral swine movement between states, including test and slaughter and control contact with domestic pigs. According to the OIE, treatment with antibiotics is not being implemented anywhere as a control measure [256]. Despite its potential application to control an outbreak at the farm level [267], antibiotic therapy is currently strongly discouraged because it does not allow the total clearance of the infection and involves the use of high doses with a considerable cost and poses some additional problems due to the limits of maximum residue in animals destined to human consumption.


  1. 1. Allsup TN. Abortion in sheep associated with Brucella abortus infection. Veterinary Record. 1969;84(5):104-108.
  2. 2. Stoenner HG. Isolation of Brucella abortus from sheep. Journal of the American Veterinary Medical Association 1951;118(887):101-102.
  3. 3. Corbel MJ. Brucellosis: an overview. Emerging Infectious Diseases. 1997;3(2):213-221; PM:9204307.
  4. 4. Refai M. Incidence and control of brucellosis in the Near East region. Veterinary Microbiology. 2002;90(1-4):81-110.
  5. 5. Samaha H, Al-Rowaily M, Khoudair RM, and Ashour HM. Multicenter study of brucellosis in Egypt. Emerging Infectious Diseases. 2008;14(12):1916-1918.
  6. 6. Zammit TA. A preliminary note on the examination of the blood of goats suffering from Mediterranean fever, Part III Proceedings of the Royal Society of London 1905; 76B:377-378.
  7. 7. Bruce D. Note on the discovery of a microorganism in Malta Fever. Practitioner. 1887;39:161-170.
  8. 8. Vassallo DJ. The corps disease: brucellosis and its historical association with the Royal Army Medical Corps. Journal of the Royal Army Medical Corps. 1992;138(3):140-150.
  9. 9. Nicoletti P. A short history of brucellosis. Veterinary Microbiology. 2002;90(1-4):5-9.
  10. 10. Plumb GE, Olsen SC, and Buttke D. Brucellosis: ‘One Health’ challenges and opportunities. Revue Scientifique et Technique (International Office of Epizootics). 2013;32(1):271-278.
  11. 11. Arroyo CI, Lopez Rodriguez MJ, Sapina AM, Lopez LA, and Sacristan AR. Probable transmission of brucellosis by breast milk. Journal of Tropical Pediatrics. 2006;52(5):380-381.
  12. 12. Kato Y, Masuda G, Itoda I, Imamura A, Ajisawa A, and Negishi M. Brucellosis in a returned traveler and his wife: probable person-to-person transmission of Brucella melitensis Journal of Travel Medicine. 2007;14(5):343-345.
  13. 13. Mantur BG, Mangalgi SS, and Mulimani M. Brucella melitensis—a sexually transmissible agent? Lancet. 1996;347(9017):1763.
  14. 14. Stantic-Pavlinic M, Cec V, and Mehle J. Brucellosis in spouses and the possibility of interhuman infection. Infection. 1983;11(6):313-314.
  15. 15. Ficht TA, and Adams LG. In: Barrett Alan DT and Stanberry LR, eds. Vaccines for Biodefense and Emerging and Neglected Diseases; 2009:807-829.
  16. 16. Pappas G, Akritidis N, Bosilkovski M, and Tsianos E. Brucellosis. New England Journal of Medicine. 2005;352(22):2325-2336.
  17. 17. Pappas G, Papadimitriou P, Akritidis N, Christou L, and Tsianos EV. The new global map of human brucellosis. Lancet Infectious Diseases. 2006;6(2):91-99.
  18. 18. Franco MP, Mulder M, Gilman RH, and Smits HL. Human brucellosis. Lancet Infectious Diseases. 2007;7(12):775-786.
  19. 19. Godfroid J, Cloeckaert A, Liautard JP, Kohler S, Fretin D, Walravens K, Garin-Bastuji B, and Letesson JJ. From the discovery of the Malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis. Veterinary Research. 2005;36(3):313-326.
  20. 20. Perkins SD, Smither SJ, and Atkins HS. Towards a Brucella vaccine for humans. FEMS Microbiology Reviews. 2010;34(3):379-394.
  21. 21. Roth F, Zinsstag J, Orkhon D, Chimed-Ochir G, Hutton G, Cosivi O, Carrin G, and Otte J. Human health benefits from livestock vaccination for brucellosis: case study. Bulletin of the World Health Organization. 2003;81(12):867-876.
  22. 22. Zinsstag J, Roth F, Orkhon D, Chimed-Ochir G, Nansalmaa M, Kolar J, and Vounatsou P. A model of animal-human brucellosis transmission in Mongolia. Preventive Veterinary Medicine. 2005;69(1-2):77-95.
  23. 23. Alton GG. In: Duncan JR and Nielsen K, eds. Animal Brucellosis. Boston; 1990:383-409.
  24. 24. Blasco JM. Control and eradication strategies for Brucella melitensis infection in sheep and goats. Prilozi. 2010;31(1):145-165.
  25. 25. FAO. Brucella melitensis in Eurasia and the Middle East. FAO Animal Production and Health Proceedings. 2009;10.
  26. 26. Nicoletti P. Brucellosis: past, present and future. Prilozi. 2010;31(1):21-32.
  27. 27. Maudlin I, and Weber-Mosdorf S. The control of neglected zoonotic diseases. WHO/SDE/FOS/2006.1. 2006.
  28. 28. McDermott J, Grace D, and Zinsstag J. Economics of brucellosis impact and control in low-income countries. Revue Scientifique et Technique (International Office of Epizootics). 2013;32(1):249-261.
  29. 29. Adone R, and Pasquali P. Epidemiosurveillance of brucellosis. Revue Scientifique et Technique (International Office of Epizootics). 2013;32(1):199-205.
  30. 30. Crespo León F. Brucelosis ovina y caprina. World Organisation for Animal Health (OIE), Paris; 1994:1-450.
  31. 31. Kolar J. Some experience from brucellosis control with Rev. 1 vaccine in a heavily infected country- Mongolia. FAO/WHO/OIE Round Table on the Use of Rev. 1 Vaccine in Small Ruminants and Cattle, Alfort, France; 1995:77-81.
  32. 32. EFSA. Porcine brucellosis (Brucella suis): scientific opinion of the Panel on Animal Health and Welfare 2009;
  33. 33. Crespo León F, Saez Llorente JL, Reviriego Gordejo FJ, Rodriguez Ferri EF, and Duran FM. Complementary tools for the control and eradication of caprine and ovine brucellosis in the European Union. Revue Scientifique et Technique (International Office of Epizootics). 2012;31(3):985-996.
  34. 34. Smits HL. Brucellosis in pastoral and confined livestock: prevention and vaccination. Revue Scientifique et Technique (International Office of Epizootics). 2013;32(1):219-228;
  35. 35. Rodriguez Ferri EF, and Crespo León F. Brucelosis. Veterinaria en Castilla y León. 2000;13:13-43.
  36. 36. Kreizinger Z, Foster JT, Ronai Z, Sulyok KM, Wehmann E, Janosi S, and Gyuranecz M. Genetic relatedness of Brucella suis biovar 2 isolates from hares, wild boars and domestic pigs. Veterinary Microbiology. 2014;172(3-4):492-498.
  37. 37. Rhyan JC, Nol P, Quance C, Gertonson A, Belfrage J, Harris L, Straka K, and Robbe-Austerman S. Transmission of brucellosis from elk to cattle and bison, Greater Yellowstone area, U.S.A., 2002-2012. Emerging Infectious Diseases. 2013;19(12):1992-1995.
  38. 38. Reviriego FJ, Moreno MA, and Dominguez L. Risk factors for brucellosis seroprevalence of sheep and goat flocks in Spain. Preventive Veterinary Medicine. 2000;44(3-4):167-173.
  39. 39. Robinson A. Guidelines for coordinated human and animal brucellosis surveillance. FAO Animal Production and Health Paper. 2003;156.
  40. 40. Diaz-Aparicio E, and Diaz. Epidemiology of brucellosis in domestic animals caused by Brucella melitensis, Brucella suis and Brucella abortus. Revue Scientifique et Technique (International Office of Epizootics). 2013;32(1):53-60.
  41. 41. Blasco JM, and Molina-Flores B. Control and eradication of Brucella melitensis infection in sheep and goats. Veterinary Clinics of North America: Food Animal Practice. 2011;27(1):95-104.
  42. 42. Alvarez J, Saez JL, Garcia N, Serrat C, Perez-Sancho M, Gonzalez S, Ortega MJ, GouJ, Carbajo L, Garrido F, et al. Management of an outbreak of brucellosis due to B. melitensis in dairy cattle in Spain. Research in Veterinary Science. 2010;90(2):208-211
  43. 43. Saez JL, Sanz C, Duran M, Garcia P, Fernandez F, Minguez O, Carbajo L, Mardones F, Perez A, Gonzalez S, et al. Comparison of depopulation and S19-RB51 vaccination strategies for control of bovine brucellosis in high prevalence areas. Veterinary Record. 2014;174(25):634.
  44. 44. Garin-Bastuji B, Blasco JM, Grayon M, and Verger JM. Brucella melitensis infection in sheep: present and future. Veterinary Research. 1998;29(3-4):255-274.
  45. 45. Nicoletti P. Further evaluations of serologic test procedures used to diagnose brucellosis. American Journal of Veterinary Research. 1969;30(10):1811-1816.
  46. 46. Kolar J. Diagnosis and control of brucellosis in small ruminants. Preventive Veterinary Medicine. 1984;2(1-4):215-225.
  47. 47. Blasco JM, Garin-Bastuji B, Marin CM, Gerbier G, Fanlo J, Jimenez de Bagues MP, and Cau C. Efficacy of different rose bengal and complement fixation antigens for the diagnosis of Brucella melitensis infection in sheep and goats. Veterinary Record. 1994;134(16):415-420.
  48. 48. Jacques I, Olivier-Bernardin V, and Dubray G. Efficacy of ELISA compared to conventional tests (RBPT and CFT) for the diagnosis of Brucella melitensis infection in sheep. Veterinary Microbiology. 1998;64(1):61-73.
  49. 49. Diaz-Aparicio E, Aragon V, Marin C, Alonso B, Font M, Moreno E, Perez-Ortiz S, Blasco JM, Diaz R, and Moriyon I. Comparative analysis of Brucella serotype A and M and Yersinia enterocolitica O:9 polysaccharides for serological diagnosis of brucellosis in cattle, sheep, and goats. Journal of Clinical Microbiology. 1993;31(12):3136-3141.
  50. 50. Nielsen K, Gall D, Smith P, Balsevicius S, Garrido F, Ferrer MD, Biancifiori F, Dajer A, Luna E, Samartino L, et al. Comparison of serological tests for the detection of ovine and caprine antibody to Brucella melitensis. Revue Scientifique et Technique (International Office of Epizootics). 2004;23(3):979-987.
  51. 51. Poester FP, Samartino LE, and Santos RL. Pathogenesis and pathobiology of brucellosis in livestock. Revue Scientifique et Technique (International Office of Epizootics). 2013;32(1):105-115.
  52. 52. Godfroid J, Nielsen K, and Saegerman C. Diagnosis of brucellosis in livestock and wildlife. Croatian Medical Journal. 2010;51(4):296-305..
  53. 53. Nielsen K. Diagnosis of brucellosis by serology. Veterinary Microbiology. 2002;90(1-4):447-459.
  54. 54. Nielsen K, Smith P, Yu W, Nicoletti P, Jungersen G, Stack J, and Godfroid J. Serological discrimination by indirect enzyme immunoassay between the antibody response to Brucella spp. and Yersinia enterocolitica O:9 in cattle and pigs. Veterinary Immunology and Immunopathology. 2006;109(1-2):69-78.
  55. 55. McGiven JA, Nicola A, Commander NJ, Duncombe L, Taylor AV, Villari S, Dainty A, Thirlwall R, Bouzelmat N, Perrett LL, et al. An evaluation of the capability of existing and novel serodiagnostic methods for porcine brucellosis to reduce false positive serological reactions. Veterinary Microbiology. 2012;160(3-4):378-386.
  56. 56. Zaccheus MV, Ali T, Cloeckaert A, Zygmunt MS, Weintraub A, Iriarte M, Moriyon I, and Widmalm G. The epitopic and structural characterization of Brucella suis biovar 2 O-polysaccharide demonstrates the existence of a new M-negative C-negative smooth Brucella serovar. PloS one. 2013;8(1):e53941.
  57. 57. Scientific Committee on Animal Health and Animal Welfare. Brucellosis in Sheep and Goats. 2001 European Commission SANCO/C.2/AII/R23/2001, 1-89.
  58. 58. Ragan V, Vroegindewey G, and Babcock S. International standards for brucellosis prevention and management. Revue Scientifique et Technique (International Office of Epizootics). 2013;32(1):189-198.
  59. 59. Oie. Bovine Brucellosis. Manual of diagnostic tests and vaccines for terrestrial animals. 2009; Chapter 2.4.3.
  60. 60. Morgan WJ, Mackinnon DJ, Lawson JR, and Cullen GA. The rose bengal plate agglutination test in the diagnosis of brucellosis. Veterinary Record. 1969;85(23):636-641.
  61. 61. OIE. Porcine brucellosis. Manual of diagnostic tests and vaccines for terrestrial animals. 2008; Chapter 2.8.5.
  62. 62. OIE. Caprine and Ovine Brucellosis (excluding Brucella ovis). Manual of diagnostic tests and vaccines for terrestrial animals. 2009; Chapter 2.7.2.
  63. 63. Abernethy DA, Menzies FD, McCullough SJ, McDowell SW, Burns KE, Watt R, Gordon AW, Greiner M, and Pfeiffer DU. Field trial of six serological tests for bovine brucellosis. Veterinary Journal. 2012;191(3):364-370.
  64. 64. Matope G, Muma JB, Toft N, Gori E, Lund A, Nielsen K, and Skjerve E. Evaluation of sensitivity and specificity of RBT, c-ELISA and fluorescence polarisation assay for diagnosis of brucellosis in cattle using latent class analysis. Veterinary Immunology and Immunopathology. 2011;141(1-2):58-63.
  65. 65. Sanogo M, Thys E, Achi YL, Fretin D, Michel P, Abatih E, Berkvens D, and Saegerman C. Bayesian estimation of the true prevalence, sensitivity and specificity of the rose bengal and indirect ELISA tests in the diagnosis of bovine brucellosis. Veterinary Journal. 2013;195(1):114-120.
  66. 66. Minas A, Stournara A, Minas M, Stack J, Petridou E, Christodoulopoulos G, and Krikelis V. Validation of a fluorescence polarization assay (FPA) performed in microplates and comparison with other tests used for diagnosing B. melitensis infection in sheep and goats. Journal of Immunological Methods. 2007;320(1-2):94-103.
  67. 67. Ramirez-Pfeiffer C, Diaz-Aparicio E, Gomez-Flores R, Rodriguez-Padilla C, Morales-Loredo A, and Alvarez-Ojeda G. Use of the Brucella melitensis native hapten to diagnose brucellosis in goats by a rapid, simple, and specific fluorescence polarization assay. Clinical and Vaccine Immunology. 2008;15(6):911-915.
  68. 68. Gall D, and Nielsen K. Serological diagnosis of bovine brucellosis: a review of test performance and cost comparison. Revue Scientifique et Technique (International Office of Epizootics). 2004;23(3):989-1002.
  69. 69. Greiner M, Verloo D, and de MF. Meta-analytical equivalence studies on diagnostic tests for bovine brucellosis allowing assessment of a test against a group of comparative tests. Preventive Veterinary Medicine. 2009;92(4):373-381.
  70. 70. Munoz PM, Marin CM, Monreal D, Gonzalez D, Garin-Bastuji B, Diaz R, Mainar-Jaime RC, Moriyon I, and Blasco JM. Efficacy of several serological tests and antigens for diagnosis of bovine brucellosis in the presence of false-positive serological results due to Yersinia enterocolitica O:9. Clinical and Diagnostic Laboratory Immunology. 2005;12(1):141-151.
  71. 71. Garcia-Bocanegra I, Allepuz A, Perez JJ, Alba A, Giovannini A, Arenas A, Candeloro L, Pacios A, Saez JL, and Gonzalez MA. Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep. Veterinary Journal. 2014;199(3):439-445.
  72. 72. Muktaderul A, Md.Ariful I, Mst.Minara K, and Byeong-Kirl B. Evaluation of four serological tests for the detection of brucellosis in goats and cattle under the field condition of Bangladesh. Asian Journal of Biological Sciences. 2011;4:477-482.
  73. 73. Rahman AK, Saegerman C, Berkvens D, Fretin D, Gani MO, Ershaduzzaman M, Ahmed MU, and Emmanuel A. Bayesian estimation of true prevalence, sensitivity and specificity of indirect ELISA, rose bengal test and slow agglutination test for the diagnosis of brucellosis in sheep and goats in Bangladesh. Preventive Veterinary Medicine. 2013;110(2):242-252.
  74. 74. Munoz PM, Blasco JM, Engel B, de Miguel MJ, Marin CM, Dieste L, and Mainar-Jaime RC. Assessment of performance of selected serological tests for diagnosing brucellosis in pigs. Veterinary Immunology and Immunopathology. 2012;146(2):150-158.
  75. 75. Praud A, Gimenez O, Zanella G, Dufour B, Pozzi N, Antras V, Meyer L, and Garin-Bastuji B. Estimation of sensitivity and specificity of five serological tests for the diagnosis of porcine brucellosis. Preventive Veterinary Medicine. 2012;104(1-2):94-100.
  76. 76. Dieste-Perez L, Blasco JM, de Miguel MJ, Moriyon I, and Munoz PM. Diagnostic performance of serological tests for swine brucellosis in the presence of false positive serological reactions. Journal of Microbiological Methods. 2015;111C:57-63.
  77. 77. Diaz-Aparicio E, Marin C, Alonso-Urmeneta B, Aragon V, Perez-Ortiz S, Pardo M, Blasco JM, Diaz R, and Moriyon I. Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats. Journal Clinical Microbiology. 1994;32(5):1159-1165.
  78. 78. Alton GG, Jones LM, Angus RD, and Verger JM. Techniques for the brucellosis laboratory. Paris, France: Inra Publications; 1988.
  79. 79. Minas A, Stournara A, Minas M, Papaioannou A, Krikelis V, and Tselepidis S. Validation of fluorescence polarization assay (FPA) and comparison with other tests used for diagnosis of B. melitensis infection in sheep. Veterinary Microbiology. 2005;111(3-4):211-221.
  80. 80. Chappel RJ, and Hayes J. Comparison of radioimmunoassay with the complement fixation test and the indirect haemolysis test in the field diagnosis of bovine brucellosis. Journal of Hygiene. 1983;90(1):67-70.
  81. 81. Blasco JM, Marin C, Jimenez de BM, Barberan M, Hernandez A, Molina L, Velasco J, Diaz R, and Moriyon I. Evaluation of allergic and serological tests for diagnosing Brucella melitensis infection in sheep. Journal Clinical Microbiology. 1994;32(8):1835-1840.
  82. 82. Garin-Bastuji B, Blasco JM, Marin C, and Albert D. The diagnosis of brucellosis in sheep and goats, old and new tools. Small Ruminant Research. 2006;62:63-70.
  83. 83. Mainar-Jaime RC, Munoz PM, de Miguel MJ, Grillo MJ, Marin CM, Moriyon I, and Blasco JM. Specificity dependence between serological tests for diagnosing bovine brucellosis in Brucella-free farms showing false positive serological reactions due to Yersinia enterocolitica O:9. Canadian Veterinary Journal. 2005;46(10):913-916.
  84. 84. Robson JM, Harrison MW, Wood RN, Tilse MH, McKay AB, and Brodribb TR. Brucellosis: re-emergence and changing epidemiology in Queensland. Medical Journal of Australia. 1993;159(3):153-158.
  85. 85. Stoffregen WC, Olsen SC, and Bricker BJ. Parenteral vaccination of domestic pigs with Brucella abortus strain RB51. American Journal of Veterinary Research. 2006;67(10):1802-1808.
  86. 86. Rogers RJ, Cook DR, Ketterer PJ, Baldock FC, Blackall PJ, and Stewart RW. An evaluation of three serological tests for antibody to Brucella suis in pigs. Australian Veterinary Journal. 1989;66(3):77-80.
  87. 87. Dieste-Perez L, Barberan M, Munoz PM, Moriyon I, and Blasco JM. Clinical and histological features of brucellin skin test responses in Brucella suis biovar 2 infected pigs. Veterinary Immunology and Immunopathology. 2015;163(1-2):77-85.
  88. 88. Perrett LL, McGiven JA, Brew SD, and Stack JA. Evaluation of competitive ELISA for detection of antibodies to Brucella infection in domestic animals. Croatian Medical Journal. 2010;51(4):314-319.
  89. 89. Carlsson HE, Hurvell B, and Lindberg AA. Enzyme-linked immunosorbent assay (ELISA) for titration of antibodies against Brucella abortus and Yersinia enterocolitica. Acta Pathologica Microbiologica Scandinavica Series C. 1976;84(3):168-176.
  90. 90. Minas A, Stournara A, Christodoulopoulos G, and Katsoulos PD. Validation of a competitive ELISA for diagnosis of Brucella melitensis infection in sheep and goats. Veterinary Journal. 2008;177(3):411-417.
  91. 91. Nielsen K, Smith P, Yu WL, Elmgren C, Halbert G, Nicoletti P, Perez B, Conde S, Samartino L, Nicola A, et al. Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals. Veterinary Immunology and Immunopathology. 2008;125(3-4):246-250.
  92. 92. Burriel AR, Christodoulopoulod G, Bisias G, and Fthenakis GC. Comparison of fluorescence polarization assay, indirect ELISA and competitive ELISA methods for diagnosis of Brucella melitensis infection in small ruminants. Small Ruminant Research.2004;54(3):243-247.
  93. 93. Nielsen KH, Kelly L, Gall D, Nicoletti P, and Kelly W. Improved competitive enzyme immunoassay for the diagnosis of bovine brucellosis. Veterinary Immunology and Immunopathology. 1995;46(3-4):285-291.
  94. 94. Paulo PS, Vigliocco AM, Ramondino RF, Marticorena D, Bissi E, Briones G, Gorchs C, Gall D, and Nielsen K. Evaluation of primary binding assays for presumptive serodiagnosis of swine brucellosis in Argentina. Clinical and Diagnostic Laboratory Immunology. 2000;7(5):828-831.
  95. 95. Fensterbank R. Brucellosis in cattle, sheep and goats: diagnosis, control and vaccination. In: Office International des Epizooties, ed. Technical Series; 1987:9-35.
  96. 96. Mahajan NK, and Kulshreshtha RC. Comparison of serological tests for Brucella melitensis infection in sheep. Tropical Animal Health and Production. 1991;23(1):11-16.
  97. 97. Waghela S, Wandera JG, and Wagner GG. Comparison of four serological tests in the diagnosis of caprine brucellosis. Research in Veterinary Science. 1980;28(2):168-171.
  98. 98. Radwan AI, Bekairi SI, Al-Mukayel AA, and Abu Agla OS. Control of ovine brucellosis in Najdi sheep in Saudi Arabia. Tropical Animal Health and Production. 1984;16(4):213-218.
  99. 99. McGiven JA, Stack JA, Perrett LL, Tucker JD, Brew SD, Stubberfield E, and MacMillan AP. Harmonisation of European tests for serological diagnosis of Brucella infection in bovines. Revue Scientifique et Technique (International Office of Epizootics). 2006;25(3):1039-1053.
  100. 100. O'Grady D, Byrne W, Kelleher P, O'Callaghan H, Kenny K, Heneghan T, Power S, Egan J, and Ryan F. A comparative assessment of culture and serology in the diagnosis of brucellosis in dairy cattle. Veterinary Journal. 2014;199(3):370-375.
  101. 101. Ferris RA, Schoenbaum MA, and Crawford RP. Comparison of serologic tests and bacteriologic culture for detection of brucellosis in swine from naturally infected herds. Journal of the American Veterinary Medical Association. 1995;207(10):1332-1333.
  102. 102. Lord VR, Cherwonogrodzky JW, Marcano MJ, and Melendez G. Serological and bacteriological study of swine brucellosis. Journal Clinical Microbiology. 1997;35(1):295-297.
  103. 103. Vanzini VR, Aguirre NP, Valentini BS, Torioni de Echaide S, Lugaresi CI, Marchesino MD, and Nielsen K. Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples. Veterinary Microbiology. 2001;82(1):55-60.
  104. 104. Jones LM, and Marly J. Titration of a Brucella protein allergen in sheep sensitized with Brucella melitensis Annals of Veterinary Research. 1975;6(2):173-178.
  105. 105. Dieste-Perez L, Blasco JM, de Miguel MJ, Marin CM, Barberan M, Conde-Alvarez R, Moriyon I, and Munoz PM. Performance of skin tests with allergens from B. melitensis B115 and rough B. abortus mutants for diagnosing swine brucellosis. Veterinary Microbiology. 2014;168(1):161-168.
  106. 106. Pouillot R, Garin-Bastuji B, Gerbier G, Coche Y, Cau C, Dufour B, and Moutou F. The brucellin skin test as a tool to discriminate false positive serological reactions in bovine brucellosis. Veterinary Research. 1997;28(4):365-374.
  107. 107. Riber U, and Jungersen G. Cell-mediated immune responses differentiate infections with Brucella suis from Yersinia enterocolitica serotype O:9 in pigs. Veterinary Immunology and Immunopathology. 2007;116(1-2):13-25.
  108. 108. Fensterbank R. Allergic diagnosis of brucellosis. In: Nijhoff, ed. Brucella melitensis;1985:167-172. Dordrecht.
  109. 109. Velasco J, Diaz R, Grillo MJ, Barberan M, Marin C, Blasco JM, and Moriyon I. Antibody and delayed-type hypersensitivity responses to Ochrobactrum anthropi cytosolic and outer membrane antigens in infections by smooth and rough Brucella spp. Clinical and Diagnostic Laboratory Immunology. 1997;4(3):279-284.
  110. 110. Jiang X, and Baldwin CL. Effects of cytokines on intracellular growth of Brucella abortus. Infection and Immunity. 1993;61(1):124-134.
  111. 111. Kim S, Lee DS, Watanabe K, Furuoka H, Suzuki H, and Watarai M. Interferon-gamma promotes abortion due to Brucella infection in pregnant mice. BMC Microbiology. 2005;5:22.
  112. 112. Vitry MA, Hanot Mambres D, De Trez C, Akira S, Ryffel B, Letesson JJ, and Muraille E. Humoral immunity and CD4+ Th1 cells are both necessary for a fully protective immune response upon secondary infection with Brucella melitensis. Journal of Immunology. 2014;192(8):3740-3752.
  113. 113. Weynants V, Godfroid J, Limbourg B, Saegerman C, and Letesson JJ. Specific bovine brucellosis diagnosis based on in vitro antigen-specific gamma interferon production. Journal Clinical Microbiology. 1995;33(3):706-712.
  114. 114. Duran-Ferrer M, Leon L, Nielsen K, Caporale V, Mendoza J, Osuna A, Perales A, Smith P, De-Frutos C, Gomez-Martin B, et al. Antibody response and antigen-specific gamma-interferon profiles of vaccinated and unvaccinated pregnant sheep experimentally infected with Brucella melitensis. Veterinary Microbiology. 2004;100(3-4):219-231.
  115. 115. Perez-Sancho M, Duran-Ferrer M, Garcia-Seco T, Macias P, Garcia N, Martinez I, Ruiz E, Legaz E, Diez-Guerrier A, Gonzalez S, et al. Interferon-gamma responses in sheep exposed to virulent and attenuated Brucella melitensis strains. Veterinary Immunology and Immunopathology. 2014;160(1-2):123-128.
  116. 116. Elberg SS, and Meyer KF. Caprine immunization against brucellosis; a summary of experiments on the isolation, properties and behaviour of a vaccine strain. Bulletin of the World Health Organization. 1958;19(4):711-724.
  117. 117. Corbel, MJ. Brucellosis in humans and animals. World Health Organization, Food and Agriculture Organization of the United Nations, and World Organization for Animal Health. World Health Organization. 2006;
  118. 118. De Miguel MJ, Marin CM, Munoz PM, Dieste L, Grillo MJ, and Blasco JM. Development of a selective culture medium for primary isolation of the main Brucella species. Journal of Clinical Microbiology. 2011;49(4):1458-1463.
  119. 119. Blasco JM. Estado Actual de la Brucelosis en España. Profesion Veterinaria.2004;15(58):22-34.
  120. 120. McGiven JA. New developments in the immunodiagnosis of brucellosis in livestock and wildlife. Revue Scientifique et Technique (International Office of Epizootics). 2013;32(1):163-176.
  121. 121. Hornitzky M, and Searson J. The relationship between the isolation of Brucella abortus and serological status of infected, non-vaccinated cattle. Australian Veterinary Journal. 1986;63(6):172-174.
  122. 122. Ferreira AC, Almendra C, Cardoso R, Pereira MS, Beja-Pereira A, Luikart G, and Correa de Sa MI. Development and evaluation of a selective medium for Brucella suis. Research in Veterinary Science. 2012;93(2):565-567.
  123. 123. Her M, Cho DH, Kang SI, Cho YS, Hwang IY, Bae YC, Yoon H, Heo YR, Jung SC, and Yoo H. The development of a selective medium for the Brucella abortus strains and its comparison with the currently recommended and used medium. Diagnostic Microbiology and Infectious Disease. 2010;67(1):15-21.
  124. 124. Yu WL, and Nielsen K. Review of Detection of Brucella spp. by Polymerase Chain Reaction. Croatian Medical Journal. 2010;51(4):306-313.
  125. 125. Nan W, Tan P, Wang Y, Xu Z, Mao K, Peng D, and Chen Y. Duplex PCR for differentiation of the vaccine strain Brucella suis S2 and B. suis biovar 1 from other strains of Brucella spp. Veterinary Journal. 2014;201(3):427-428.
  126. 126. Kang SI, Lee SE, Kim JY, Lee K, Kim JW, Lee HK, Sung SR, Heo YR, Jung SC, and Her M. A new Brucella canis species-specific PCR assay for the diagnosis of canine brucellosis. Comparative Immunology, Microbiology and Infectious diseases. 2014;37(4):237-241.
  127. 127. Gopaul KK, Sells J, Lee R, Beckstrom-Sternberg SM, Foster JT, and Whatmore AM. Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species. BMC research notes. 2014.
  128. 128. Hinic V, Brodard I, Thomann A, Cvetnic Z, Makaya PV, Frey J, and Abril C. Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems. Journal of Microbiological Methods. 2008;75(2):375-378.
  129. 129. Bounaadja L, Albert D, Chenais B, Henault S, Zygmunt MS, Poliak S, and Garin-Bastuji B. Real-time PCR for identification of Brucella spp.: a comparative study of IS 711, bcsp31 and per target genes. Veterinary Microbiology. 2009;137(1-2):156-164.
  130. 130. Garcia-Yoldi D, Marin CM, de Miguel MJ, Munoz PM, Vizmanos JL, and Lopez-Goni I. Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clinical Chemistry. 2006;52(4):779-781.
  131. 131. Huber B, Scholz HC, Lucero N, and Busse HJ. Development of a PCR assay for typing and subtyping of Brucella species. International Journal of Medical Microbiology. 2009;299(8):563-573.
  132. 132. Mayer-Scholl A, Draeger A, Gollner C, Scholz HC, and Nockler K. Advancement of a multiplex PCR for the differentiation of all currently described Brucella species. Journal of Microbiological Methods. 2010;80(1):112-114.
  133. 133. Mirnejad R, Doust RH, Kachuei R, Mortazavi SM, Khoobdel M, and Ahamadi A. Simultaneous detection and differentiates of Brucella abortus and Brucella melitensis by combinatorial PCR. Asian Pacific Journal of Tropical Medicine. 2012;5(1):24-28.
  134. 134. Rantakokko-Jalava K, and Jalava J. Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR. Journal of Clinical Microbiology. 2002;40(11):4211-4217.
  135. 135. Bricker BJ. PCR as a diagnostic tool for brucellosis. Veterinary Microbiology. 2002;90(1-4):435-446.
  136. 136. O'Leary S, Sheahan M, and Sweeney T. Brucella abortus detection by PCR assay in blood, milk and lymph tissue of serologically positive cows. Research in Veterinary Science. 2006;81(2):170-176.
  137. 137. Tomaso H, Kattar M, Eickhoff M, Wernery U, Al DS, Straube E, Neubauer H, and Scholz HC. Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR. BMC Infectious Diseases. 2010;10:100.
  138. 138. Godfroid J, Scholz HC, Barbier T, Nicolas C, Wattiau P, Fretin D, What more AM, Cloeckaert A, Blasco JM, Moriyon I, et al. Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century. Preventive Veterinary Medicine. 2011;102(2):118-131.
  139. 139. Olsen SC. Recent developments in livestock and wildlife brucellosis vaccination. Revue Scientifique et Technique (International Office of Epizootics). 2013;32(1):207-217.
  140. 140. Moriyon I, Grillo MJ, Monreal D, Gonzalez D, Marin C, Lopez-Goni I, Mainar-Jaime RC, Moreno E, and Blasco JM. Rough vaccines in animal brucellosis: structural and genetic basis and present status. Veterinary Research. 2004;35(1):1-38.
  141. 141. Elzer PH, Hagius SD, Davis DS, DelVecchio VG, and Enright FM. Characterization of the caprine model for ruminant brucellosis. Veterinary Microbiology. 2002;90(1-4):425-431.
  142. 142. Bosseray N. Brucella melitensis Rev. 1 living attenuated vaccine: stability of markers, residual virulence and immunogenicity in mice. Biologicals: Journal of the International Association of Biological Standardization. 1991;19(4):355-363.
  143. 143. Elberg SS, and Meyer KF. Caprine immunization against brucellosis; a summary of experiments on the isolation, properties and behaviour of a vaccine strain. Bulletin of the World Health Organization. 1958;19(4):711-724.
  144. 144. Zundel E, Verger JM, Grayon M, and Michel R. Conjunctival vaccination of pregnant ewes and goats with Brucella melitensis Rev 1 vaccine: safety and serological responses. Annals of Veterinary Research. 1992;23(2):177-188.
  145. 145. Thomas EL, Bracewell CD, and Corbel MJ. Characterisation of Brucella abortus strain 19 cultures isolated from vaccinated cattle. Veterinary Record. 1981;108(5):90-93.
  146. 146. Jimenez de Bagues MP, Marin CM, Barberan M, and Blasco JM. Responses of ewes to B. melitensis Rev1 vaccine administered by subcutaneous or conjunctival routes at different stages of pregnancy. Annals of Veterinary Research. 1989;20(2):205-213.
  147. 147. Pacheco WA, Genovez ME, Pozzi CR, Silva MP, Azevedo SS, Did CC, Piatti RM, Pinheiro ES, Castro V, Miyashiro S, et al. Excretion of Brucella abortus vaccine B19 strain during a reproductive cycle in dairy cows. Brazilian Journal of Microbiology. 2012:594-601.
  148. 148. Jones LM, and Marly J. Serological and bacteriological studies of ewes vaccinated with Brucella melitensis strain Rev. 1 during lactation. Annals of Veterinary Research. 1975;6(1):67-71.
  149. 149. Alton GG. Duration of the immunity produced in goats by the Rev. 1 Brucella melitensis vaccine. Journal of Comparative Pathology. 1966;76(3):241-253.
  150. 150. Alton GG. Further studies on the duration of the immunity produced in goats by the Rev. 1 Brucella melitensis vaccine. Journal of Comparative Pathology. 1968;78(2):173-178.
  151. 151. Lord VR, Schurig GG, Cherwonogrodzky JW, Marcano MJ, and Melendez GE. Field study of vaccination of cattle with Brucella abortus strains RB51 and 19 under high and low disease prevalence. American Journal of Veterinary Research. 1998;59(8):1016-1020.
  152. 152. Diaz-Aparicio E, Hernandez L, and Suarez-Guemes F. Protection against brucellosis in goats, five years after vaccination with reduced-dose Brucella melitensis Rev 1 vaccine. Tropical Animal Health and Production. 2004;36(2):117-121.
  153. 153. Nicoletti P. In: Nielsen K and Duncan JR, eds. Animal Brucellosis. Boca Raton: CRC Press; 1990:248-299.
  154. 154. Scharp DW, al Khalaf SA, al Muhanna MW, Cheema RA, and Godana W. Use of mass vaccination with a reduced dose of REV 1 vaccine for Brucella melitensis control in a population of small ruminants. Tropical Animal Health and Production. 1999;31(3):135-141.
  155. 155. Beckett FW, and MacDiarmid SC. The effect of reduced-dose Brucella abortus strain 19 vaccination in accredited dairy herds. British Veterinary Journal. 1985;141(5):507-514.
  156. 156. Blasco JM. A review of the use of B. melitensis Rev 1 vaccine in adult sheep and goats. Preventive Veterinary Medicine. 1997;31(3-4):275-283.
  157. 157. Crowther RW, Orphanides A, and Polydorou K. Vaccination of adult sheep with reduced doses of Brucella melitensis strain Rev. 1: safety and serological responses. Tropical Animal Health and Production. 1977;9(2):85-91.
  158. 158. Confer AW, Hall SM, Faulkner CB, Espe BH, Deyoe BL, Morton RJ, and Smith RA. Effects of challenge dose on the clinical and immune responses of cattle vaccinated with reduced doses of Brucella abortus strain 19. Veterinary Microbiology. 1985;10(6):561-575.
  159. 159. Woodard LF, and Jasman RL. Comparative efficacy of an experimental S45/20 bacterin and a reduced dose of strain 19 vaccine against bovine brucellosis. American Journal of Veterinary Research. 1983;44(5):907-910.
  160. 160. al-Khalaf SA, Mohamad BT, and Nicoletti P. Control of brucellosis in Kuwait by vaccination of cattle, sheep and goats with Brucella abortus strain 19 or Brucella melitensis strain Rev. 1. Tropical Animal Health and Production. 1992;24(1):45-49.
  161. 161. Al-Shamakh IH. A brief review of the national project for the control of brucellosis in the Kingdom of Saudi Arabia. FAO/WHO/OIE Round Table, on the use of Rev.-1 vaccine in small ruminants and cattle. CNVA Alfort, France, September 1995.
  162. 162. Nicoletti P. A preliminary report on the efficacy of adult cattle vaccination using Strain 19 in selected dairy herds in Florida. Proceedings of Annual Meeting of United States of Animal Health Association. 197680):91-106.
  163. 163. Nicoletti P. The effects of adult cattle vaccination with strain 19 on the incidence of brucellosis in dairy herds in Florida and Puerto Rico. Proceedings of the Annual Meeting of the United States Animal Health Association. 1979(83):75-80.
  164. 164. Fensterbank R, and Plommet M. Vaccination against bovine brucellosis with a low dose of strain 19 administered by the conjunctival route. IV. Comparison between two methods of vaccination. Annals of Veterinary Research. 1979;10(1):131-139.
  165. 165. Ali Kojouri G, and Gholami M. Post vaccination follow-up of Brucella melitensis in blood stream of sheep by PCR assay. Comparative Clinical Pathology. 2009;18(4):439-442.
  166. 166. Banai M, Abramson M, Mayer I, Chechick K, Hoida G, Zamir O, Bardenstein S, Cohen A, and Davidson M. Problems associated with the persistence and possible horizontal transfer of Brucella melitensis Rev. I vaccine in connection with serological surveillance in Israel. FAO/WHO/OIE Round Table on the use of Rev. 1 vaccine in small ruminants an cattle CNEVA Alfort, France; 1995:69-76.
  167. 167. Sales Henriques HLR, Hueston WD, Hoblet KH, and Shulaw WP. Field trials evaluating the safety and serologic reactions of reduced-dose Brucella melitensis Rev 1 vaccination in adult sheep. Preventive Veterinary Medicine. 1992;13(3):205-215.
  168. 168. Stournara A, Minas A, Bourtzi-Chatzopoulou E, Stack J, Koptopoulos G, Petridou E, and Sarris K. Assessment of serological response of young and adult sheep to conjunctival vaccination with Rev-1 vaccine by fluorescence polarization assay (FPA) and other serological tests for B. melitensis. Veterinary Microbiology. 2007;119(1):53-64.
  169. 169. Lantier F and Fensterbank R. Kinetics of Rev1 infection in sheep. In: Verger JM and Plommet M, eds. Brucella melitensis. Martinus Nijhoff, Dordrecht; 1985:247-251.
  170. 170. Tolari F, and Salvi G. Segnalazione di un caso di orchite bilaterale in un capretto in sequito a vaccinatzione con Rev. 1. Estrato dagli Anm Faculta Med Vet. 1980:33.
  171. 171. Corbel MJ, Stuart FA, Brewer RA, Jeffrey M, and Bradley R. Arthropathy associated with Brucella abortus strain 19 vaccination in cattle. II. Experimental studies. British Veterinary Journal. 1989;145(4):347-355.
  172. 172. Olsen SC, and Stoffregen WS. Essential role of vaccines in brucellosis control and eradication programs for livestock. Expert Review of Vaccines. 2005;4(6):915-928.
  173. 173. Banai M. Control of small ruminant brucellosis by use of Brucella melitensis Rev. 1 vaccine: laboratory aspects and field observations. Veterinary Microbiology. 2002;90(1-4):497-519.
  174. 174. Blasco JM, and Diaz R. Brucella melitensis Rev-1 vaccine as a cause of human brucellosis. Lancet. 1993;342(8874):805.
  175. 175. Meyer ME. Characterization of Brucella abortus strain 19 isolated from human and bovine tissues and fluids. American Journal of Veterinary Research. 1985;46(4):902-904.
  176. 176. Pivnick H, Worton H, Smith DL, and Barnum D. Infection of veterinarians in Ontario by Brucella abortus strain 19. Canadian journal of public healthRevue canadienne de sante publique. 1966;57(5):225-231.
  177. 177. Vincent P, Joubert L, and Prave M. Two occupational cases of brucellar infection after inoculation of B 19 vaccine. Bulletin de l'Academie Veterinaire de France. 1970;43(2):89-97.
  178. 178. Grillo MJ, Bosseray N, and Blasco JM. In vitro markers and biological activity in mice of seed lot strains and commercial Brucella melitensis Rev 1 and Brucella abortus B19 vaccines. Biologicals: Journal of the International Association of Biological Standardization. 2000;28(2):119-127.
  179. 179. Alton GG. In: Verger J, and Plommet M eds. Brucella melitensis Dordrecht/Boston/Lancaster: Martinus Nijhoff; 1985:215-227.
  180. 180. Mukherjee F, Jain J, Grillo MJ, Blasco JM, and Nair M. Evaluation of Brucella abortus S19 vaccine strains by bacteriological tests, molecular analysis of ery loci and virulence in BALB/c mice. Biologicals: Journal of the International Association of Biological Standardization. 2005;33(3):153-160.
  181. 181. Menzies PI. Vaccination programs for reproductive disorders of small ruminants. Animal Reproduction Science. 2012;130(3-4):162-172.
  182. 182. Garcia-Yoldi D, Le FP, Marin CM, de Miguel MJ, Munoz PM, Vergnaud G, and Lopez-Goni I. Assessment of genetic stability of Brucella melitensis Rev 1 vaccine strain by multiple-locus variable-number tandem repeat analysis. Vaccine. 2007;25(15):2858-2862.
  183. 183. Alton GG. Rev. 1 Brucella melitensis vaccine. Serological reactions in Maltese goats. Journal of Comparative Pathology. 1967;77(3):327-329.
  184. 184. Alton GG, Fensterbank R, and Plommet AM. La brucellose de la chevre. In: Yvor P and Perrin G eds. Les maladies de la chevre. 1984.
  185. 185. Herr S, Ehret WJ, Ribeiro LM, and Chaparro F. The persistence of serological reactions following heifer vaccination with Brucella abortus strain 19 on brucellosis-free farms. Journal of the South African Veterinary Association. 1990;61(1):11-13.
  186. 186. Plommet M, and Plommet AM. Vaccination against bovine brucellosis with a low dose of strain 19 administered by the conjunctival route. Annals of Veterinary Research. 1976;7(1):1-8.
  187. 187. Fensterbank R, Pardon P, and Marly J. Comparison between subcutaneous and conjunctival route of vaccination with Rev. 1 strain against Brucella melitensis infection in ewes. Annals of Veterinary Research. 1982;13(4):295-301.
  188. 188. Verger JM, Grayon M, Zundel E, Lechopier P, and Olivier-Bernardin V. Comparison of the efficacy of Brucella suis strain 2 and Brucella melitensis Rev. 1 live vaccines against a Brucella melitensis experimental infection in pregnant ewes. Vaccine. 1995;13(2):191-196.
  189. 189. Delgado S, Fernandez M, and C rmenes P. Influence of age and stage of gestation on serological response to subcutaneous or conjunctival Brucella melitensis strain Rev. 1 vaccination in ewes. Small Ruminant Research. 1996;19(1):63-68.
  190. 190. McGiven JA, Tucker JD, Perrett LL, Stack JA, Brew SD, and MacMillan AP. Validation of FPA and cELISA for the detection of antibodies to Brucella abortus in cattle sera and comparison to SAT, CFT, and iELISA. Journal of Immunological Methods. 2003;278(1-2):171-178.
  191. 191. Nielsen K. The serological response of cattle immunized with Yersinia enterocolitica O:9 or O:16 to Yersinia and Brucella abortus antigens in enzyme immunoassays. Veterinary Immunology and Immunopathology. 1990;24(4):373-382.
  192. 192. Debbarh HS, Cloeckaert A, Zygmunt MS, and Dubray G. Identification of sero-reactive Brucella melitensis cytosoluble proteins which discriminate between antibodies elicited by infection and Rev. 1 vaccination in sheep. Veterinary Microbiology. 1995;44(1):37-48.
  193. 193. Cloeckaert A, Baucheron S, Vizcaino N, and Zygmunt MS. Use of recombinant BP26 protein in serological diagnosis of Brucella melitensis infection in sheep. Clinical and Diagnostic Laboratory Immunology 2001;8(4):772-775.
  194. 194. Salih-Alj DH, Cloeckaert A, Bezard G, Dubray G, and Zygmunt MS. Enzyme-linked immunosorbent assay with partially purified cytosoluble 28-kilodalton protein for serological differentiation between Brucella melitensis-infected and B. melitensis Rev. 1-vaccinated sheep. Clinical and Diagnostic Laboratory Immunology. 1996;3(3): 305-308.
  195. 195. Debbarh HS, Zygmunt MS, Dubray G, and Cloeckaert A. Competitive enzyme-linked immunosorbent assay using monoclonal antibodies to the Brucella melitensis BP26 protein to evaluate antibody responses in infected and B. melitensis Rev. 1 vaccinated sheep. Veterinary Microbiology. 1996;53(3-4):325-337.
  196. 196. Xin T, Yang H, Wang N, Wang F, Zhao P, Wang H, Mao K, Zhu H, and Ding J. Limitations of the BP26 protein-based indirect enzyme-linked immunosorbent assay for diagnosis of Brucellosis. Clinical and Vaccine Immunology: CVI. 2013;20(9):1410-1417.
  197. 197. Ciocchini AE, Serantes DA, Melli LJ, Guidolin LS, Iwashkiw JA, Elena S, Franco C, Nicola AM, Feldman MF, Comerci DJ, et al. A bacterial engineered glycoprotein as a novel antigen for diagnosis of bovine brucellosis. Veterinary Microbiology. 2014;172(3-4):455-465.
  198. 198. Baldi PC, Giambartolomei GH, Goldbaum FA, Abdon LF, Velikovsky CA, Kittelberger R, and Fossati CA. Humoral immune response against lipopolysaccharide and cytoplasmic proteins of Brucella abortus in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype O:9. Clinical and Diagnostic Laboratory Immunology. 1996;3(4):472-476.
  199. 199. Pajuaba AC, Silva DA, and Mineo JR. Evaluation of indirect enzyme-linked immunosorbent assays and IgG avidity assays using a protein A-peroxidase conjugate for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Clinical and Vaccine Immunology: CVI. 2010;17(4):588-595.
  200. 200. Blasco JM, Diaz R, Moriyon I, and Salvo MD. Evaluation of a radial immunodiffusion test for diagnosing brucellosis in sheep and its possible value for differentiating infected from Brucella melitensis Rev 1 vaccinated sheep. Developments in Biological Standardization. 1984;56:507-511.
  201. 201. Diaz R, Garatea P, Jones LM, and Moriyon I. Radial immunodiffusion test with a Brucella polysaccharide antigen for differentiating infected from vaccinated cattle. Journal of Clinical Microbiology. 1979;10(1):37-41.
  202. 202. Kittelberger R, Reichel MP, Joyce MA, and Staak C. Serological crossreactivity between Brucella abortus and Yersinia enterocolitica O:9. III. Specificity of the in vitro antigen-specific gamma interferon test for bovine brucellosis diagnosis in experimentally Yersinia enterocolitica 0:9-infected cattle. Veterinary Microbiology. 1997;57(4):361-371.
  203. 203. Jimenez de Bagues MP, Marin CM, Blasco JM, Moriyon I, and Gamazo C. An ELISA with Brucella lipopolysaccharide antigen for the diagnosis of B. melitensis infection in sheep and for the evaluation of serological responses following subcutaneous or conjunctival B. melitensis strain Rev 1 vaccination. Veterinary Microbiology. 1992;30(2-3):233-241.
  204. 204. Jones LM, Berman DT, Moreno E, Deyoe BL, Gilsdorf MJ, Huber JD, and Nicoletti P. Evaluation of a radial immunodiffusion test with polysaccharide B antigen for diagnosis of bovine brucellosis. Journal of Clinical Microbiology. 1980;12(6):753-760.
  205. 205. Cosivi O, and Corbel MJ. WHO consultation on the development of new/improved brucellosis vaccines. 17 December 1997, Geneva, Switzerland. Biologicals: Journal of the International Association of Biological Standardization. 1998;26(4):361-363.
  206. 206. Gonzalez D, Grillo MJ, de Miguel MJ, Ali T, Arce-Gorvel V, Delrue RM, Conde-Alvarez R, Munoz P, Lopez-Goñi I, Iriarte M, et al. Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export. PLoS One. 2008;3(7):e2760.
  207. 207. Cloeckaert A, Jacques I, de WP, Dubray G, and Limet JN. Protection against Brucella melitensis or Brucella abortus in mice with immunoglobulin G (IgG), IgA, and IgM monoclonal antibodies specific for a common epitope shared by the Brucella A and M smooth lipopolysaccharides. Infections and Immunity. 1992;60(1):312-315.
  208. 208. Limet J, Plommet AM, Dubray G, and Plommet M. Immunity conferred upon mice by anti-LPS monoclonal antibodies in murine brucellosis. Annales de l' Institute Pasteur Immunology. 1987;138(3):417-424.
  209. 209. Montaraz JA, Winter AJ, Hunter DM, Sowa BA, Wu AM, and Adams LG. Protection against Brucella abortus in mice with O-polysaccharide-specific monoclonal antibodies. Infections and Immunity. 1986;51(3):961-963.
  210. 210. Adone R, Francia M, Pistoia C, Petrucci P, Pesciaroli M, and Pasquali P. Protective role of antibodies induced by Brucella melitensis B115 against B. melitensis and Brucella abortus infections in mice. Vaccine. 2012;30(27):3992-3995.
  211. 211. Barrio MB, Grillo MJ, Munoz PM, Jacques I, Gonzalez D, de Miguel MJ, Marin CM, Barberan M, Letesson JJ, Gorvel JP, et al. Rough mutants defective in core and O-polysaccharide synthesis and export induce antibodies reacting in an indirect ELISA with smooth lipopolysaccharide and are less effective than Rev 1 vaccine against Brucella melitensis infection of sheep. Vaccine. 2009;27(11):1741-1749.
  212. 212. Adone R, Francia M, and Ciuchini F. Brucella melitensis B115-based complement fixation test to detect antibodies induced by Brucella rough strains. Journal of Applied Microbiology. 2008;105(2):567-574.
  213. 213. Ivanov AV, Salmakov KM, Olsen SC, and Plumb GE. A live vaccine from Brucella abortus strain 82 for control of cattle brucellosis in the Russian Federation. Animal Health Research Reviews. 2011;12(1):113-121.
  214. 214. Nicoletti P. Vaccination against Brucella. Advances in Biotechnology Processes. 1990;13:147-168.
  215. 215. Schurig GG, Roop RM, Bagchi T, Boyle S, Buhrman D, and Sriranganathan N. Biological properties of RB51; a stable rough strain of Brucella abortus. Veterinary Microbiology. 1991;28(2):171-188.
  216. 216. Ugalde JE, Comerci DJ, Leguizamon MS, and Ugalde RA. Evaluation of Brucella abortus phosphoglucomutase (pgm) mutant as a new live rough-phenotype vaccine. Infection and Immunity. 2003;71(11):6264-6269.
  217. 217. Stevens MG, Hennager SG, Olsen SC, and Cheville NF. Serologic responses in diagnostic tests for brucellosis in cattle vaccinated with Brucella abortus 19 or RB51. Journal of Clinical Microbiology. 1994;32(4):1065-1066.
  218. 218. Uza FA, Samartino L, Schurig G, Carrasco A, Nielsen K, Cabrera RF, and Taddeo HR. Effect of vaccination with Brucella abortus strain RB51 on heifers and pregnant cattle. Veterinary Research Communications. 2000;24(3):143-151.
  219. 219. Cloeckaert A, Zygmunt MS, and Guilloteau LA. Brucella abortus vaccine strain RB51 produces low levels of M-like O-antigen. Vaccine. 2002;20(13-14):1820-1822.
  220. 220. Olsen SC, Evans D, Hennager SG, Cheville NF, and Stevens MG. Serologic responses of Brucella abortus strain 19 calfhood-vaccinated cattle following adult vaccination with strain RB51. Journal of Veterinary Diagnostic Investigation. 1996;8(4):451-454.
  221. 221. Palmer MV, Cheville NF, and Jensen AE. Experimental infection of pregnant cattle with the vaccine candidate Brucella abortus strain RB51: pathologic, bacteriologic, and serologic findings. Veterinary Pathology. 1996;33(6):682-691.
  222. 222. Palmer MV, Olsen SC, and Cheville NF. Safety and immunogenicity of Brucella abortus strain RB51 vaccine in pregnant cattle. American Journal of Veterinary Research. 1997;58(5):472-477.
  223. 223. Van Metre DC, Kennedy GA, Olsen SC, Hansen GR, and Ewalt DR. Brucellosis induced by RB51 vaccine in a pregnant heifer. Journal of American Veterinary Medicine Association. 1999;215(10):1491-1493.
  224. 224. Cheville NF, Olsen SC, Jensen AE, Stevens MG, Palmer MV, and Florance AM. Effects of age at vaccination on efficacy of Brucella abortus strain RB51 to protect cattle against brucellosis. American Journal of Veterinary Research. 1996;57(8):1153-1156.
  225. 225. Olsen SC. Immune responses and efficacy after administration of a commercial Brucella abortus strain RB51 vaccine to cattle. Veterinary Therapeutics. 2000;1(3):183-191.
  226. 226. Lord VR, Cherwonogrodzky JW, Schurig GG, Lord RD, Marcano MJ, and Melendez GE. Venezuelan field trials of vaccines against brucellosis in swine. American Journal of Veterinary Research. 1998;59(5):546-551.
  227. 227. el Idrissi AH, Benkirane A, el MM, Bouslikhane M, Berrada J, and Zerouali A. Comparison of the efficacy of Brucella abortus strain RB51 and Brucella melitensis Rev. 1 live vaccines against experimental infection with Brucella melitensis in pregnant ewes. Revue Scientifique et Technique (International Office of Epizootics). 2001;20(3):741-747.
  228. 228. Herrera E, Rivera A, Palomares EG, Hernandez-Castro R, and Diaz-Aparicio E. Isolation of Brucella melitensis from a RB51-vaccinated seronegative goat. Tropical Animal Health and Production. 2011;43(6):1069-1070.
  229. 229. Martínez DI, Morales JA, Peniche AE, Molina B RMA, Loeza R, Robledo ML, Morales JF, and Flores-Castro R. Use of RB51 vaccine for small ruminants brucellosis prevention in Veracruz, Mexico. International Journal of Diary Sciences. 2010;5(1):10-17.
  230. 230. Adone R, Ciuchini F, Marianelli C, Tarantino M, Pistoia C, Marcon G, Petrucci P, Francia M, Riccardi G, and Pasquali P. Protective properties of rifampin-resistant rough mutants of Brucella melitensis. Infection and Immunity. 2005;73(7):4198-4204.
  231. 231. Adone R, Francia M, and Ciuchini F. Evaluation of Brucella melitensis B115 as rough-phenotype vaccine against B. melitensis and B. ovis infections. Vaccine. 2008;26(38):4913-4917.
  232. 232. Winter AJ, Schurig GG, Boyle SM, Sriranganathan N, Bevins JS, Enright FM, Elzer PH, and Kopec JD. Protection of BALB/c mice against homologous and heterologous species of Brucella by rough strain vaccines derived from Brucella melitensis and Brucella suis biovar 4. American Journal of Veterinary Research. 1996;57(5):677-683.
  233. 233. Stoffregen WC, Johnson CS, and Olsen SC. Immunogenicity and safety of a natural rough mutant of Brucella suis as a vaccine for swine. Research in Veterinary Science. 2013;95(2):451-458.
  234. 234. Schumaker B. Risks of Brucella abortus spillover in the Greater Yellowstone area. Revue Scientifique et Technique. 2013;32(1):71-77.
  235. 235. European Commission. Health and Consumer Directoriate-General. Task force on monitoring animal disease eradication (2009). Working document on eradication of bovine, sheep and goats brucellosis of the Task force on monitoring animal disease eradication in the EU. SANCO/6095/2009.
  236. 236. Manthei CA. In: Association USLSS ed. 63rd Annual Meeting, US Live Stock Sanitary Association.1959:91-97.
  237. 237. Erasmus JA, and Erasmus MC. The use of reduced-dose Brucella abortus strain 19 vaccine in the control of bovine brucellosis. Journal of the South African Veterinary Association. 1987;58(2):71-75.
  238. 238. Sanz C, Saez JL, Alvarez J, Cortes M, Pereira G, Reyes A, Rubio F, Martin J, Garcia N, Dominguez L, et al. Mass vaccination as a complementary tool in the control of a severe outbreak of bovine brucellosis due to Brucella abortus in Extremadura, Spain. Preventive Veterinary Medicine. 2010;97(2):119-125.
  239. 239. Olsen SC, Bricker B, Palmer MV, Jensen AE, and Cheville NF. Responses of cattle to two dosages of Brucella abortus strain RB51: serology, clearance and efficacy. Research in Veterinary Science. 1999;66(2):101-105.
  240. 240. Poester FP, Goncalves VS, Paixao TA, Santos RL, Olsen SC, Schurig GG, and Lage AP. Efficacy of strain RB51 vaccine in heifers against experimental brucellosis. Vaccine. 2006;24(25):5327-5334.
  241. 241. Martins H, Garin-Bastuji B, Lima F, Flor L, Pina FA, and Boinas F. Eradication of bovine brucellosis in the Azores, Portugal—outcome of a 5-year programme (2002-2007) based on test-and-slaughter and RB51 vaccination. Preventive Veterinary Medicine. 2009;90(1-2):80-89.
  242. 242. Blasco JM, and Moriyon I. Eradication of bovine brucellosis in the Azores, Portugal--outcome of a 5-year programme (2002-2007) based on test-and-slaughter and RB51 vaccination. Preventive Veterinary Medicine. 2010;94(1-2):154-157.
  243. 243. Samartino LE, Fort M, Gregoret R, and Schurig GG. Use of Brucella abortus vaccine strain RB51 in pregnant cows after calfhood vaccination with strain 19 in Argentina. Preventive Veterinary Medicine. 2000;45(3-4):193-199.
  244. 244. Bercovich Z. Maintenance of Brucella abortus-free herds: a review with emphasis on the epidemiology and the problems in diagnosing brucellosis in areas of low prevalence. Veterinary Quarterly. 1998;20(3):81-88.
  245. 245. Benkirane A. Ovine and caprine brucellosis: world distribution and control/eradication strategies in West Asia/North Africa region. Small Ruminant Research. 2006;62:19-25.
  246. 246. Racloz V, Schelling E, Chitnis N, Roth F, and Zinsstag J. Persistence of brucellosis in pastoral systems. Revue Scientifique et Technique (International Office of Epizootics). 2013;32(1):61-70.
  247. 247. European Commission. Health and Consumer Directoriate-General.Task force on monitoring animal disease, e. (2009). Working document on eradication of bovine, sheep and goats brucellosis of the Task force on monitoring animal disease eradication in the EU.SANCO/6095/2009.
  248. 248. Adone R, Francia M, Pistoia C, Pesciaroli M, and Pasquali P. B. melitensis rough strain B115 is protective against heterologous Brucella spp. infections. Vaccine. 2011;29(14):2523-2529..
  249. 249. Avila-Calderon ED, Lopez-Merino A, Jain N, Peralta H, Lopez-Villegas EO, Sriranganathan N, Boyle SM, Witonsky S, and Contreras-Rodriguez A. Characterization of outer membrane vesicles from Brucella melitensis and protection induced in mice. Clinical and Developmental Immunology. 2012:352-493.
  250. 250. Edmonds MD, Cloeckaert A, Hagius SD, Samartino LE, Fulton WT, Walker JV, Enright FM, Booth NJ, and Elzer PH. Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Deltaomp25 deletion mutant. Research in Veterinary Science. 2002;72(3):235-239.
  251. 251. Magnani DM, Harms JS, Durward MA, and Splitter GA. Nondividing but metabolically active gamma-irradiated Brucella melitensis is protective against virulent B. melitensis challenge in mice. Infection and Immunity. 2009;77(11):5181-5189.
  252. 252. Sislema-Egas F, Cespedes S, Fernandez P, Retamal-Diaz A, Saez D, and Onate A. Evaluation of protective effect of DNA vaccines encoding the BAB1_0263 and BAB1_0278 open reading frames of Brucella abortus in BALB/c mice. Vaccine. 2012;30(50):7286-7291
  253. 253. Tibor A, Jacques I, Guilloteau L, Verger JM, Grayon M, Wansard V, and Letesson JJ. Effect of P39 gene deletion in live Brucella vaccine strains on residual virulence and protective activity in mice. Infection and Immunity. 1998;66(11):5561-5564.
  254. 254. Yang X, Hudson M, Walters N, Bargatze RF, and Pascual DW. Selection of protective epitopes for Brucella melitensis by DNA vaccination. Infection and Immunity. 2005;73(11):7297-7303.
  255. 255. Perez-Sancho M, Adone R, Garcia-Seco T, Tarantino M, Diez-Guerrier A, Drumo R, Francia M, Dominguez L, Pasquali P, and Alvarez J. Evaluation of the immunogenicity and safety of Brucella melitensis B115 vaccination in pregnant sheep. Vaccine. 2014;32(16):1877-1881.
  256. 256. WAHID—OIE World Animal Health Information System;
  257. 257. Deqiu S, Donglou X, and Jiming Y. Epidemiology and control of brucellosis in China. Veterinary Microbiology. 2002;90(1-4):165-182.
  258. 258. Alton G. Brucella melitensis. In: Animal Brucellosis, Nielsen KH, and Duncan JR, eds. CRC Press, Boca Raton, Florida, USA; 1990:383-409.
  259. 259. Iowa State University. Porcine and Rangiferine Brucellosis: Brucella suis. Enzootic abortion, Contagious Abortion. Undulant Fever. The Centre of Food Security and Public Health. Iowa State University. 2009;
  260. 260. Animal and Plant Health Inspection Service, United States Department of Agriculture. Swine Brucellosis. Control/Eradication. State-Federal-Industry Uniform Methods and Rules; 1998.
  261. 261. Grupo de Trabajo sobre Prevención, control y Erradicación de la Brucelosis en América Latina y el Caribe. Programa Regional para la Prevención, Control, y Erradicación de la Brucelosis en América Latina y el Caribe. Instituto Panamericano de Protección de Alimentos y Zoonosis Programa de Salud Pública Veterinaria. 1994.
  262. 262. Resolución 63/2013. Créase el “Registro Nacional de Establecimientos Oficialmente Libres de Brucelosis Porcina”. Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA);
  263. 263. Eales KM, Norton RE, and Ketheesan N. Brucellosis in northern Australia. American Journal of Tropical Medicine and Hygiene. 2010;83(4):876-878.
  264. 264. Department of Agriculture and Fisheries. Brucella suis.
  265. 265. Althouse GC, and Rossow K. The potential risk of infectious disease dissemination via artificial insemination in swine. Reproduction in Domestic Animals. 2011;46 Suppl 2:64-67.
  266. 266. Stoffregen WC, Olsen SC, Jack WC, Bricker BJ, Palmer MV, Jensen AE, Halling SM, and Alt DP. Diagnostic characterization of a feral swine herd enzootically infected with Brucella. Journal of Veterinary Diagnostic and Investigation. 2007;19(3):227-237.
  267. 267. Dieste-Perez L, Fraile L, de Miguel MJ, Barberan M, Blasco JM, and Muñoz PM. Studies on a suitable antibiotic therapy for treating swine brucellosis. Journal of Veterinary Pharmacology and Therapeutics. 2014.

Written By

Marta Pérez-Sancho, Teresa García-Seco, Lucas Domínguez and Julio Álvarez

Submitted: December 19th, 2014 Reviewed: July 14th, 2015 Published: November 19th, 2015