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Diagnosis of Brucellosis by Serology Klaus Nielsen* Animal Diseases Research Institute, 3851 Fallow®Eld Road, Nepean, Ont., Canada K2H 8P9

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Diagnosis of Brucellosis by Serology Klaus Nielsen* Animal Diseases Research Institute, 3851 Fallow®Eld Road, Nepean, Ont., Canada K2H 8P9 Veterinary Microbiology 90 (2002) 447±459 Diagnosis of brucellosis by serology Klaus Nielsen* Animal Diseases Research Institute, 3851 Fallow®eld Road, Nepean, Ont., Canada K2H 8P9 Abstract Serological diagnosis of brucellosis began more than 100 years ago with a simple agglutination test. It was realized that this type of test was susceptible to false positive reactions resulting from,for instance,exposure to cross reacting microorganisms. It was also realized that this test format was inexpensive,simple and could be rapid,although results were subjectively scored. Therefore,a number of modi®cations were developed along with other types of tests. This served two purposes: one was to establish a rapid screening test with high sensitivity and perhaps less speci®city and a con®rmatory test,usually more complicated but also more speci®c,to be used on sera that reacted positively in screening tests. This led to another problem: if a panel of tests were performed and they did not all agree,which interpretation was correct? This problem was further compounded by the extensive use of a vaccine which gave rise to an antibody response similar to that resulting from ®eld infection. This led to the development of an assay that could distinguish vaccinal antibody,starting with precipitin tests. These tests did not perform well,giving rise to the development of primary binding assays. These assays,including the competitive enzyme immunoassay and the ¯uorescence polarization assay are at the apex of current development,providing high sensitivity and speci®city as well as speed and mobility in the case of the ¯uorescence polarization assay. Crown Copyright # 2002 Published by Elsevier Science B.V. All rights reserved. Keywords: Serology; Brucellosis; Diagnostic; Agglutination tests; Complement ®xation; Precipitation tests; Binding assay; Antigen 1. Introduction Brucellosis may be diagnosed de®nitively by isolation and identi®cation of the causative organism. This was ®rst reported by Bruce and co-workers in 1887 when they isolated Brucella melitensis from military personnel in Malta (Bruce,1887,1893 ). The disease was derived from infected goats. Brucella abortus was isolated from aborting cattle by Bang (Bang,1897 ) while B. suis was ®rst described by Taum (1914). The above three species of * Tel.: 1-613-228-6698; fax: 1-613-228-6667. E-mail address: [email protected] (K. Nielsen). 0378-1135/02/$ ± see front matter. Crown Copyright # 2002 Published by Elsevier Science B.V.All rights reserved. PII: S 0378-1135(02)00229-8 448 K. Nielsen / Veterinary Microbiology 90 (2002) 447±459 Brucella comprise the most important species which contain O-polysaccharide (OPS) on the cell surface. The OPS which is part of the lipopolysaccharide (LPS) molecule has been chemically de®ned: homopolymer of 4,6-dideoxy-4-formamide-alpha-D-mannose,linked via 1,2-glycosidic linkages (Bundle et al.,1987 ). Other important species which contain no measurable OPS on the cell surface are B. ovis,isolated by MacFarlane et al. (1952) and B. canis described by Kimberling et al. (1966). The three main species containing OPS are diagnosed serologically using either a whole cell antigen or smooth lipopolysaccharide (SLPS) prepared by chemical extraction while B. ovis and B. canis are mainly diagnosed using rough lipopolysaccharide (RLPS) or protein antigens (Blasco,1990 ). Of other Brucella species, B. neotoma,found in the desert wood rat,although diagnosed infre- quently and Brucella species of marine mammals (without of®cial species designation) may be detected serologically using B. abortus antigen (Nielsen et al.,2001 ). Because common epitopes are present in B. abortus, B. melitensis and B. suis SLPS,virtually all serological tests for antibody to these bacteria utilize B. abortus antigen (OIE Manual, 2000a,b) while the rough Brucella species are diagnosed using antigens from each species (Blasco,1990; Carmichael,1990 ). Since the majority of diagnostic tests use B. abortus antigens,these tests will be the main focus. Serology for human brucellosis is not discussed although in general,the same tests are applicable to animal and human serology. Before describing the various serological tests,it is useful to examine what is being measured. Again,the antibody response to B. abortus in cattle will be used as the example as it has been studied in most detail. The antibody response to B. abortus in cattle consists of an early IgM isotype response,the timing of which depends on the route of exposure,the dose of bacteria and the health status of the animal (Beh,1973,1974; Allan et al.,1976 ). The IgM response is followed almost immediately by production of IgG1 antibody and later by small amounts of IgG2 and IgA (Corbel,1972; Beh,1974; Allan et al.,1976; Levieux,1978a; Nielsen et al.,1984 ). Most cross reacting antibody,that is antibody resulting from exposure to microorganisms other than Brucella sp. or environmental antigens,consists mainly of IgM ( Corbel,1985 ). Serological tests that measure IgM are therefore not desirable as false positive results occur,leading to low assay speci®city. Since IgG2 and IgA antibodies accumulate later after exposure and are usually present in small and inconsistent amounts,the main isotype for serological testing is IgG1 ( Allan et al., 1976; Lamb et al.,1979; Nielsen et al.,1984; Butler et al.,1986 ). Therefore,assays that predominately measure IgG1 are the most useful. Serological diagnosis was somewhat confounded by the development of a vaccine, B. abortus S19,which was serologically indistinguishable from virulent strains ( Buck, 1930). This vaccine was and is an important aspect of the control and eradication programs for brucellosis and it was and is therefore widely used,however,before the development of primary binding assays,serological distinction was not easy,leading to allowances for higher antibody levels and a common practice of vaccinating calves before the age of 10 months (allowing for a reduction in antibody level prior to sexual maturity). These problems were largely overcome with the development of competitive enzyme immu- noassays,(CELISA) and a ¯uorescence polarization assay (FPA),both of which eliminated nearly all residual vaccinal antibody (Nielsen et al.,1995,1996 ),and the development of a vaccine, B. abortus RB51,which contains no (or minute amounts of) OPS on its cell surface (Schurig et al.,1991 ). Acceptance of these serological tests has been slow and the K. Nielsen / Veterinary Microbiology 90 (2002) 447±459 449 use of RB51 is only starting to become widespread. This has led to innumerable diagnostic dilemmas and the quarantine or slaughter of many non-infected animals. 2. Overview of serological tests 2.1. Common in-use tests Since 1897,a considerable number of serological tests have been developed. A number of these tests were modi®ed in various ways to increase performance. The list below is not all inclusive,however,it includes most of the in-use tests and some that were not widely used but interesting. Each test will be discussed brie¯y. Agglutination tests: standard tube (SAT); acidified antigen (RBT,card,BPAT); high salt; EDTA; reducing agents (2ME,DTT); rivanol (RIV); heat; antiglobulin tests; milk ring test (MRT). Complement fixation test: warm (CFT); cold; indirect hemolysis test (IHLT); hemolysis in gel (HIG). Precipitation tests: agar gel immunodiffusion test (AGID); single radial immunodiffusion test (SRD). Primary binding assays: radioimmunoassay (RIA); particle concentration fluorescence immunoassay (PCFIA); indirect enzyme immunoassay (IELISA); competitive enzyme immunoassay (CELISA); fluorescence polarization assay (FPA). 2.2. Agglutination tests Wright and Smith (1897) described the ®rst serological test for brucellosis. This test consisted of adding Brucella cells to diluted serum and observing the pattern of the cell pellet after incubation. This is very similar to the standard tube agglutination test still in use in some countries. IgM isotype of antibody is the most active agglutinin at a neutral or slightly below neutral pH (Rice and Boyes,1971; Corbel,1972; Nielsen et al.,1984 ). Therefore the tube agglutination test is susceptible to false positive reactions by cross 450 K. Nielsen / Veterinary Microbiology 90 (2002) 447±459 reacting antibody. As a result,the tube agglutination test should not be used as a diagnostic test and its discontinuation is recommended by the OIE (OIE Manual,2000a ). Because of the speci®city problems associated with the original tube agglutination test,a large number of modi®cations were made to destroy or inactivate IgM agglutinins. Of these modi®cations,the acidi®ed antigen,rivanol precipitation and the use of 2-mercaptoethanol are in common use in various laboratories,while most of the other modi®cations (heating the serum,addition of antiglobulin,antigen with increased sodium chloride,addition of chelating agents) were of local interest and are not discussed. 2.3. Acidified antigen modifications There are two commonly used tests,the rose-bengal/card test (RBT) ( Nicoletti,1967; Morgan et al.,1969; Davis,1971 ) and the buffered antigen plate agglutination test (BPAT) (Angus and Barton,1984 ). The RBT uses B. abortus S99 or S1119.3 whole cells,stained with rose-bengal while the BPAT uses B. abortus S1119.3 whole cells,stained with crystal violet and brilliant green dyes. Both antigens are used at a pH of 3.65. The low pH prevents some agglutination by IgM and encourages agglutination by IgG1 thereby reducing non- speci®c interactions (Corbel,1972,1973; Allan et al.,1976 ). Both tests are standardized, simple to perform and inexpensive. Both tests are considered suitable for screening individual animals,however,false negative reactions occur,mostly due to prozoning. Antibody resulting from B. abortus S19 vaccination and some cross reacting antibodies are detected by these tests and it is necessary to use other test(s) to con®rm reactor animals as infected. The BPAT is an OIE prescribed test for bovine,porcine,ovine and caprine brucellosis (OIE Manual, 2000a,b,d).
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