Diagnostic Approaches For Bovine Brucellosis

Overview of Brucellosis:

Brucellosis, also known as “contagious abortion’’; “Bang’s disease”, is a zoonotic and a highly contagious bacterial disease caused by several Brucella species. Each species of Brucella have a propensity to be associated with a specific host. Cattle are the common hosts for Brucella abortus; at times, disease can be caused by Brucella melitensis and Brucella suis. Brucella spp. are Gram-negative, non-capsulated, non-motile, do not produce spores and are partially acid-fast cocco-bacilli. The prime clinical symptom of brucellosis is associated with the reproductive tract and the most characteristic manifestation in pregnant animals is delayed abortion, delivery of sick calves, reduced fertility and reduced milk yield, which leads to substantial economic losses.

Considering the damage and its profound threat to humans, specific identification is essential to control the disease in animals and subsequently in humans. The diagnostic procedures for the identification of brucellosis include direct examination (tests) (microbiological analysis, DNA detection by PCR, i.e. polymerase chain reaction) and indirect examination (tests) (used either in vitro, predominantly to blood and milk or in vivo also called allergic test) (Fig. 1)

Fig.1: Direct and indirect tests for diagnosing brucellosis


Direct tests

Staining: Presumptive identification of brucellosis is accomplished by the microscopic inspection of stained smears from aborted fetuses or placentas and vaginal swabs, making use of modified Ziehl-Neelsen (stamp) staining. However, care must be taken as morphologically-comparable microbes, viz., Chlamydophila abortus, Chlamydia psittaci, and Coxiella burnetti can delude the detection due to their apparent resemblance. Brucella are Gram-negative, singly arranged short rods, show resistance to de-colorization by weak acids and stain red against a blue background in Modified Ziehl- Neelsen stain.

Culture:  Isolating bacterial culture from the samples is a prerequisite for bio-typing of strains. B. abortus can be isolated from samples, including aborted fetuses, fetal membranes, vaginal secretions, colostrum, milk, oropharyngeal lymph nodes and genital, spleen, and mammary glands. Farrell’s medium, Thayer Martin’s, Triptcase soy agar, Tryptic soy agar, Tryptone soya agar and Bacto tryptose agar are commonly used media for isolating Brucella.Colony morphology, staining and biochemical profiling (catalase, urease, and oxidase tests) form the basis of identification. The colonies of Brucella spp. are transparent, elevated, and convex with intact borders.

Molecular methods: Polymerase chain reaction (PCR) is a very promising method of diagnosis and is rapidly replacing the conventional assays. The first polymerase chain reaction (PCR) based test for brucellosis aimed at a gene coding a 43-KDa Omp from Brucella abortus strain. Numbers of nucleic acid sequences have been targeted for developing Brucella genus-specific PCR assays. A number of conventional and multiplex PCR assays hve been described by various reseachers. The first and the foremost species-specific multiplex polymerase chain reaction (PCR) known as AMOS-PCR assay was employed for the identification and differentiation of Brucella abortus biovars 1, 2, and 4, Brucella ovis, Brucella melitensis and Brucella suis biovar 1. In addition, Bruce Ladder multiplex PCR assay is able to differentiate almost all species and vaccine strain of Brucella spp. Recently, Real time-PCR assays are employed to detect B. abortus, B. suis and B. melitensis and are more rapid and sensitive than the conventional PCR assays.

Indirect test

Serum agglutination test (SAT):  Also known as slow agglutination of Wright (SAW) or slow tube agglutination test on serum is based on the principle of detecting agglutinin antibodies predominantly of IgM isotype aimed against Brucella spp. At an optimal concentration of antigen and antibodies, large antigen-antibody complexes are formed and get agglutinated at the bottom of the test tube. This reaction requires an overnight incubation at 37°C, which makes it a slow process.

Despite being a standardized and exceptionally robust test for brucellosis, lack of sensitivity and specificity are considered to be significant drawbacks of this test. The specificity of this test can be enhanced by treating serum with EDTA (chelating agent), which minimizes the cross-reactions because of IgM. This test is no longer promoted by OIE-World Organization for Animal Health for bovine brucellosis detection, but still is extensively utilized for diagnosing human brucellosis.

Rose Bengal plate (RBPT) test: RBPT is a fast screening test with >99% sensitivity. In this, B. abortus cells are stained with Rose Bengal and are placed in a buffer, which when admixed with the right amount of serum, gives a final pH of 3.65. At this pH, RBPT prevents the agglutination with IgM and detects IgG1 only. Agglutination must appear within 4 minutes for RBT, as more extended incubation periods sometimes give false results due to the generation of fibrin clots. Wherever possible, serum indicating a positive result should always be affirmed by a more specific test. However, the drawback of this test is that sometimes antibodies generated due to Brucella vaccine can also be detected giving false positive reactions.  

Complement fixation test (CFT): It detects specific antibodies belonging to IgM and IgG1 types, capable of activating complement. Basic CFT consists of incubation of Brucella abortus antigen with the dilutions of heat-inactivated serum and a titrated source of complement. After an apt period, a pre-titrated quantity of sensitized sheep erythrocytes covered with the rabbit antibody is added. The formation of primary immune complex owing to the existence of antibody isotypes in serum activates the complement. It hence is not accessible to the secondary immune complex of sheep erythrocytes and rabbit antibody, hence results in slight disintegration of erythrocytes. On the other hand, absence of a primary immune complex results in the disintegration or lysis of sheep erythrocytes by the complement.

Despite high sensitivity and specificity of CFT, it is however a laborious method and requires highly trained technicians and appropriate laboratory facilities which makes it less acceptable in developing countries. CFT detects antibodies of the IgG1 type, as antibodies of the IgM type are partially lysed during in-activation. As standardization of this test is laborious, it is gradually being replaced by ELISAs.

Milk Ring Test: In this, colored Brucella whole-cell antigen is mixed with the fresh bulk milk. The presence of anti-Brucella antibodies is confirmed by the formation of antigen-antibody complexes, which results in the formation of a purple ring on the surface of the cream. On the contrary, if the anti-Brucella antibodies are absent, the cream remains uncolored. This test is, however, not considered to be a sensitive one but can be repeated owing to its low cost.

2-Mercaptoethanol test: It is a confirmatory test for selective quantification of IgG, as disulfide bridges of IgM are converted to monomeric units, and, hence, are not capable of agglutinating. IgG is quantified by using 2-mercaptoethanol or dithiothreitol, the latter is preferred due to the high toxic levels of 2-mercaptoethanol.

Coombs test: Also known as the anti-globulin test, the Coombs test is one of the most sensitive serological tests, utilized to validate serum agglutination test (SAT) results from those animals that have given negative or ambiguous results. It detects uncompleted antibodies of IgG type that combines with the cellular antigens but fails to produce an agglutination reaction. However, this test is not recommended to test the vaccinated animal, which is the major limitation of this test.

ELISA:  The ELISA test is highly sensitive and specific, simple to execute, and require minimum equipments which are readily available from numerous commercial sources in kit form. ELISA is generally categorized into two classes; indirect ELISA (i-ELISA), and competitive ELISA (c-ELISA). Most i-ELISA uses purified lipo-polysaccharides as antigen and mainly detects IgGs or IgG sub-classes. I-ELISA is highly sensitive, but they are mostly subjected to some non-specific reactions, mainly ascribed to Yersinia infection, which further led to the development of more specific c-ELISA. c-ELISA utilizes monoclonal antibodies aimed against the specific epitopes (not shared with lipo-polysaccharides of Yersinia LPS) present in the O-chain of smooth lipo-polysaccharides of Brucella.

Fluorescence Polarization Assay (FPA): It is based on the principle that the rate of rotation of a molecule in solution and its size are inversely proportional. The molecules of bigger size (antigen-antibody complex) spin slowly and depolarize a polarized light to a lesser degree. On the other hand, molecules of smaller size (antigen only) spin faster and depolarize more. The degree of depolarization is measured in mP (milli-polarization) units.

The specific antigen of Brucella abortus tagged with fluorescein iso-thiocyanate is incubated with serum samples, and the rate of rotation is computed by employing polarized light. The presence of antibodies against Brucella spp. is confirmed by the formation of large fluorescent complexes, while the antigen remains un-complexed in the negative samples. These antigens spin at a higher speed and result in greater depolarization of light compared to those samples which are favorable for Brucella spp.

Skin test: It is an allergic test that utilizes a protein antigen “brucellergene” from Brucella to detect the specific cellular immune response incited by Brucella spp. infection. The administration of this antigen (brucellergene) is followed by local inflammation in sensitized animals. This test has a very high specificity and is useful in eliminating the false positive serological reactions, usually in animals which are latently-infected and lack any quantifiable antibody.


Brucellosis is the most common zoonotic disease worldwide, accountable for abortion, retained fetal membrane, endometritis, orchitis, and epididymitis in animals. The presence of brucellosis can be detected by several direct and indirect tests, although each method has limitations. However, no single test is applicable in any epidemiological situation and it is always recommended to use a combination of tests for accurate diagnosis of disease.

Paviter Kaur and Manisha Parmar

Department of Veterinary Microbiology, GADVASU, Ludhiana