Method

Strengths

Limitations

Recommendations

Indirect methods

Serology

Rose Bengal Plate Test (RBPT)

· Highly sensitive, inexpensive, simple, and rapid to perform.

· Detects mainly IgM and requires essential laboratory equipment and expertise.

· Low specificity, false-negative and false-positive results and not recommended for diagnosing chronic brucellosis.

· Applicable as a rapid diagnostic test.

· Confirmation of results is required, using either CFT or ELISA.

Indirect Enzyme-linked immunosorbent assay (iELISA)

· High sensitivity, is affordable, has shorter run times and requires less interpretation training.

· More sensitive than RBT. More sensitive and specific than SAT

· Measures various antibody titers (IgG, IgM, and IgA).

· Low specificity, lower than RBT.

· Appropriate in diagnosing various stages of infection, chronic brucellosis cases, and detecting incomplete antibodies.

· Ideal for screening purposes.

Competitive Enzyme-linked immunosorbent assay (cELISA)

· High specificity.

· Lower sensitivity than iELISA and species unspecific.

· Applicable for various animal species. Can process poor quality samples such as hemolysed blood and use for the confirmation of brucellosis.

Complement fixation test (CFT)

· Very specific, detecting IgM and IgG1 antibodies.

· Detects incomplete antibodies, as well as the slightest changes in antibody titres.

· False-negative results were recorded when IgG2 antibodies impede complement fixation.

· Not appropriate in detecting recent infections in cattle.

· Requires expertise for interpretation.

· The quality of its results is affected by the sample quality and the standardisation of the antigen.

· It is fitting for control and surveillance programs.

· It is used together with RBT as a confirmatory test.

Slow Agglutination Test (SAT)

· Designed mainly to detect IgM.

· Low sensitivity and specificity, slow and not advisable for use in individual animals.

· Not applicable for the detection of chronic brucellosis

· Suitable in detecting brucellosis on a herd basis.

· Use in routine diagnosis, complemented with Brucella Coombs test.

Slow agglutination of Wright with EDTA (SAW-EDTA)

· Sensitive, designed for the detection of IgM.

· Laborious and time-consuming.

· It has been replaced by slide, plate and card agglutination tests.

Lateral flow assay (LFA)

· Sensitive and specific, as sensitive as the RBT but having a much higher specificity.

· Can distinguish between IgM and IgG antibodies.

· More accurate and specific than the SAT in chronic and complex cases.

· Not suitable for large-scale screening.

· Appropriate for rapid field or bedside testing in endemic areas and where laboratories lack modern facilities.

· Suitable in the confirmation of RBT results.

· It can be used by smallholder herds to screen for and remove infected cattle or to reject milk from infected cattle.

Direct detection

Cellular test

Delayed hypersensitivity test for brucellin (DHTB)

· High specificity in non-vaccinated cattle. It detects antigens before the circulating antibodies appear and are more specific than RBT and CFT.

· Lower specificity in vaccinated cattle. Low sensitivity and may sensitise cattle that have not been exposed to the infection.

· Results should be combined with serology for confirmation at herd levels before the slaughter of infected cattle.

Culture

· The gold standard provides the definitive diagnosis of the disease with good specificity.

· It requires biosafety level 3 precautions, takes a minimum of 4 - 5 days to grow in culture and is costly.

· Sensitivity is problematic in cases where the fever is intermittent.

· In chronic disease, culture results may be negative due to bacterial eradication without complete clinical recovery.

· Application is not feasible in the routine diagnosis of the disease.

· Useful in research for identifying the species in circulation.

Polymerase chain reaction (PCR)

· Provides rapid detection and confirmation of Brucella with high specificity and low cost.

· It is more sensitive and safer than blood culture and more specific than serologic methods in diagnosing acute disease.

· The PCR-ELISA molecular method is more sensitive than other methods, with a semi-quantitative ability.

· Sensitivity and accuracy are dependent on the DNA extraction method and the quality of extracted genomic DNA.

· Challenges are faced in standardising extraction methods; infrastructure, equipment and expertise are still lacking.

· It has a lower sensitivity than ELISA, and sensitivity can also be reduced in chronic brucellosis.

· Its complexity contributes to the limited application in routine laboratory practice.

· It is applicable in assessing the treatment efficacy, species differentiation and biotyping of isolates and is convenient for diagnosing human brucellosis.

· The combined use of PCR and ELISA diagnostic tests can improve and overcome limitations in diagnosing brucellosis.

Sequencing

· It gives an insight into the genetic bases for host preference, pathogenesis, virulence, biotype differences and phylogenetic relationships.

· Enables the identification of potential targets for the development of vaccines and diagnostics to prevent and control brucellosis.

· Equipment is expensive, not readily available, and requires trained staff to carry out the procedures.

· Applicable for research on genetic variability, geographical distribution and host preferences.