brucellosis (Brucella abortus)

Secondary hosts play a small part if any in the maintenance or spread of a particular Brucella species. Brucella abortus mainly infects cattle and is the main cause of contagious abortion in cattle (Manthei and Carter, 1950; Dekeijzer, 1981; Crawford et al., 1990). However, sheep, goats, dogs, camels, buffaloes as well as feral animals may also contract B. abortus infections.

Although sheep do not easily become infected with B. abortus (Collier and Molello, 1964; Allsup, 1974) they may become carriers and excrete Brucellae for up to 40 months once they have acquired the infection (Luchsinger and Anderson, 1967). The low prevalence of naturally acquired B. abortus infections reported in goats makes this animal species irrelevant as a host for B. abortus (Mathur, 1967).

Isolation of B. abortus from swine (Ray, 1979), horses (Robertson et al., 1973), and camels (Al-Khalaf and El-Khaladi, 1989) in areas with enzootic brucellosis clearly indicates that these species may acquire infection with B. abortus. However, their significance as a host for B. abortus is doubtful, as these animal species usually do not intermingle with cattle. Dogs with naturally acquired B. abortus infections play an important role in the epidemiology of cattle brucellosis (Chary, 1970). The relationship between infected dogs and outbreaks of brucellosis in cattle has not only been reported but has also been demonstrated (Prior, 1976; Forbes, 1990).

Studies indicate that feral animal such as buffalo, swine, deer, fox, hare and rodents are susceptible to Brucellae. However, their role as a host for Brucella particularly in intensive cattle farming is not clearly established (Cook et al., 1966; Moore and Schnurrenberger, 1981; Schnurrenberger et al., 1985). In fact, data compiled during brucellosis control and eradication campaigns suggest that brucellosis disappears from wildlife when it is eradicated from domestic animals (Ray, 1979).

The significance of fowl as a reservoir of Brucella is unclear (Anezykowski, 1972). The role of small feral animals is also not clear although studies of experimentally induced and naturally occurring Brucella-infection in flies, arthropods and other parasites suggest that they may be susceptible to infection with Brucella (Thorpe et al., 1965; Britov et al., 1979).

Data on the prevalence of brucellosis and the success or failure to control or eradicate the disease are published in a great number of scientific papers. However, a substantial number of those publications are now only of historical value since eradication programs employed in various developed countries have successfully eliminated B. abortus infection from cattle.

Re-appearance of the disease in brucellosis-free areas is usually limited and the infected herds are immediately slaughtered, to prevent spread of brucellosis. Therefore, reports on the prevalence of brucellosis do not always appear in the literature. It usually depends on the frequency of surveillance for brucellosis in each country, and whether the results are published. Therefore, the literature references given in the geographical distribution tables are not always up to date and countries where no information is given are not always free from brucellosis. For current information on disease incidence, see OIE's WAHID Interface.

During 2011, 18 countries reported outbreaks of brucellosis to AU-IBAR recording a total of 1066 outbreaks, 136,987 cases and 709 deaths (AU-IBAR, 2011). The highest number of outbreaks was reported by Algeria (367), followed by South Africa (282) and Egypt (165). Uganda reported the highest number of cases (136,987) followed by Egypt (1120) and Algeria (1019).

Countries reporting brucellosis to AU-IBAR 

NS=Not specified

Clinical signs

An outbreak of brucellosis is hardly ever confined to one animal and there are no pathognomonic signs. Therefore, clinical examination of aborted material is not of great diagnostic value. Demonstration of characteristic clumps of Brucella organisms in stained smears of hygroma fluid, chorionic epithelium, or the use of fluorescent antibody techniques to examine foetal stomach content and uterine, or vaginal exudate may provide a tentative diagnosis (Corbel, 1973).

Bacteriological examination

Bacteriological examination of lochia of aborting cattle is the method of choice for diagnosing early infections (Erasmus, 1986). However, the procedure is laborious, time consuming, costly and cannot routinely be used as a diagnostic procedure in developed or developing countries. Moreover, the probability of successful recovery of B. abortus is strongly reduced when the material is heavily contaminated and negative culture results do not exclude infection.

Serological tests

Body fluids such as serum, uterine discharge, vaginal mucus, milk, or semen plasma from suspected cattle may contain different quantities of antibodies of the M, G₁, G₂, and A types directed against Brucella (Beh, 1974). Because infected cattle may or may not produce all antibody types in detectable quantities several tests are used to detect brucellosis. The commonly used tests are the milk ring test (MRT), serum agglutination test (SAT), complement fixation test (CFT), Rose Bengal (RB) plate test, anti-globulin (Coombs) test, 2-mercaptoethanol, rivanol and the enzyme-linked immunosorbent assay (ELISA). The use of several tests to reliably detect brucellosis suggests shortcomings in each of the tests.

The milk ring test (MRT) is cheap, easy, simple and quick to perform. It detects lacteal anti-Brucella IgM and IgA bound to milk fat globules. However, it tests false positive when milk that contains colostrum, milk at the end of the lactation period, milk from cows suffering from a hormonal disorder or milk from cows with mastitis are tested (Bercovich and Moerman, 1979). Milk that contains low concentrations of lacteal IgM and IgA or which is lacking the fat-clustering factors tests false-negative (Keer et al., 1959; Tanwani and Pathak, 1971; Patterson and Deyoe, 1978). Because lacteal antibodies rapidly decline after abortion or parturition, the reliability of the MRT, using 1 ml milk, to detect Brucella antibodies in individual cattle or in tank milk is strongly reduced (Hill, 1966). Although the MRT performed with 8-ml milk improved the detection of brucellosis in tank milk (Bercovich and Lagendijk, 1978), it may test false positive when traces of colostrum are present in tank milk (Bercovich and Moerman, 1979).

The serum agglutination test (SAT), which historically has been the principal serological test used to detect brucellosis, measures agglutinating antibodies of the IgM, IgG₁, IgG_2, and IgA types (Levieux, 1974). The SAT is relatively simple and easy to perform but it requires basic laboratory equipment. It can be used to detect acute infections, as antibodies of the IgM type usually appear first after infection and are more reactive in the SAT than antibodies of the IgG₁ and IgG₂ types (Beh, 1974; Levieux, 1974). However, because the SAT may yield both false-negative or false-positive results (Corbel et al., 1984) it effectively detects brucellosis only on a herd basis.

The complement fixation test (CFT) detects specific antibodies of the IgM and IgG₁ type that fix complement (Hill, 1963a; Levieux, 1974). The CFT is highly specific (Hill, 1963b) but it is laborious and requires highly trained personnel as well as suitable laboratory facilities. This makes the CFT less suitable for use in developing countries. Although its specificity is very important for the control and eradication of brucellosis it may test false negative when antibodies of the IgG₂ type hinder complement fixation (MacMillan, 1990). The CFT measures more antibodies of the IgG₁ type than antibodies of the IgM type, as the latter are partially destroyed during inactivation. Since antibodies of the IgG₁ type usually appear after antibodies of the IgM type, control and surveillance for brucellosis is best done with SAT and CFT (EU directive 64/432/EC (F2) AnnexA. II, and C).

The Rose Bengal plate test is a spot agglutination technique. Because the test does not need special laboratory facilities and is simple and easy to perform it is used to screen sera for Brucella antibodies. The test detects specific antibodies of the IgM and IgG types and is more effective in detecting antibodies of the IgG₁ type than IgM and IgG₂ types (Levieux, 1974). The test may yield negative results in infected cattle that give positive results with the CFT (Rose and Roepke, 1957). Although the low pH (+3.6) of the antigen enhances the specificity of the test, the temperature of the antigen and the ambient temperature at which the reaction takes place may influence the sensitivity and specificity of the RB test (MacMillan, 1990).

The anti-globulin (Coombs) test detects (incomplete Brucella) antibodies of the IgG₂ type and is used to confirm SAT results (Hill, 1963b). The Coombs test, although laborious, is particularly important when the SAT is positive and CFT results are negative or inconclusive (Kiss, 1971). However, Coombs test results are indicative for infection only when its titres are at least two times the titres of the SAT (Hill, 1963b). This is the test's main limitation, as not all infected cattle show this ratio. The 2-mercaptoethanol and the rivanol tests detect specific IgG (Rossi and Cantini, 1969) and are usually used to differentiate between infected and vaccinated cattle.

Nielsen et al. (1981) reviewed several ELISA procedures and the antigens, conjugates and substrates that can be used in the assay. The ELISA has proven to be specific and as sensitive as the MRT and SAT in detecting Brucella antibodies in milk and serum. ELISA results are usually also in agreement with CFT results (Ruppanner and Taaijke, 1980; Stemshorn et al., 1980; Bercovich and Taaijke, 1990). The test can be used for screening and confirmation of brucellosis in both milk and serum (Bercovich and Taaijke, 1990). However, depending on the presence of traces of colostrum in the milk, or the presence of low concentrations of lacteal immunoglobulin, the ELISA may test false positive or false negative (Bercovich and Taaijke, 1990; Kerkhofs et al., 1990). It seems that the ELISA is less sensitive than the CFT, as some infected cattle that test positive with the CFT may test negative with the ELISA (Sutherland, 1984; Cargill et al., 1985). Some researchers imply that the main advantage of the ELISA when compared with the CFT lies in its relative simple test procedure (Sutherland et al., 1986). The assay is very costly when only a few samples are tested, therefore it is unsuitable for testing individual animals but is the ideal test for screening suspected herds.

Since the reliability of serological tests to detect brucellosis depends on antibodies that may or may not be present at the time of examination, inevitably some infected animals may elude detection. Because the skin-delayed-type-hypersensitivity (SDTH) test is independent of circulating antibodies it should be added to the serological tests to improve detection of brucellosis. The SDTH test confirms serologic test results, confirms brucellosis in cattle with ambivalent serologic test results and detects latent carriers of Brucella. Furthermore, the SDTH test does not sensitize cattle for several consecutive SDTH tests (Bercovich, 1999). Therefore, the SDTH test should be the test of choice in developing countries, as cattle in those countries are usually not tagged so that serological test results could be related to the individual animal. Where the animals are tagged a combined use of the SAT and SDTH tests increase the reliability of brucellosis diagnosis (Bercovich, 1999).

Prolonged treatment of infected domestic animals with a high dosage of antibiotics is not used due to the appearance of antibiotics in the human food chain and its interference with the production of milk products. Moreover, as Brucellae are facultative intracellular bacteria, relapses after treatment usually occur. Therefore, efforts are directed at prevention or eradication of brucellosis.

Legislation is needed to effectively control and eradicate brucellosis. There are various combinations of diagnostic tests that can be used to detect cattle infected with B. abortus in tagged animals. Untagged animals can repeatedly (every 5-6 weeks) be tested with the SDTH test, as the test does not sensitize cattle for subsequent SDTH tests (Bercovich et al., 1992). Suspect herds must be tested at regular intervals until all animals test negative. Animals that test positive should be removed from the herd.

In areas with endemic brucellosis only vaccination will control brucellosis. Vaccination reduces the number of infected animals and eventually permits disease control. Brucella vaccines in use are the live B. abortus Strain-19 vaccine and to a lesser extent the whole-cell-killed adjuvant B. abortus 45/20 vaccine. In the 1990s, a new B. abortus vaccine RB51 has been introduced.

Vaccination with 40-120 x 10⁹ CFU (classical dose) of living Brucella abortus Strain-19 gives a fair to good protection but it also has some disadvantages (Plommet, 1991). It may cause abortion in pregnant cattle and/or induce an antibody response that confuses the serological diagnosis of brucellosis for 12-36 months. It is excreted in the milk and may induce brucellosis in humans. To diminish these undesirable effects of vaccination with S-19, two vaccination procedures have been suggested. In one procedure calves are vaccinated once with 3-10 x 10⁹ CFU (‘reduced dose’) at an age of 4-8 month and for the second time with 3-10 x 10⁹ CFU as adults. The second procedure suggests a conjunctival vaccination of calves with two drops of vaccine containing 4-10 x 10⁹ CFU at an age of 4-10 months and a second conjunctival vaccination with the same dose six months later.

Brucella abortus strain RB51 used for vaccination was selected by growth of B. abortus strain 2308 in the presence of rifampicin. The protective effect of this vaccine in cattle is similar to that of S-19. Compared with S-19 B. abortus RB51 vaccine causes less abortion (Cheville et al., 1996) and does not induce production of agglutinating antibodies of the IgM type, although specific IgG is produced (Stevens and Olsen, 1996). Depending on the doses used it may cause placentitis that leads to pre-term expulsion of the foetus. The vaccine has been approved for use in the USA to allow additional data on filed use under controlled conditions (Information Circular, WHO Mediterranean Zoonoses Control Center).

The use of Brucella abortus 45/20 vaccine is less common than S-19 because in comparison to S-19 it does not give lasting immunity. The vaccine does not induce detectable agglutinating antibodies and is not harmful but it gives a marked skin reaction on the injection site. Two initial vaccinations at specific intervals and an annual booster are needed for good protection (Plommet, 1991).