Development and Evaluation of Indirect Enzyme-Linked Immunosorbent Assays for Diagnosis of Meliodosis in Marine Mammals at Ocean Park
Melioidosis, an important disease of humans in the tropics, is caused by the gram-negative organism Burkholderia pseudomallei. Deaths due to melioidosis in marine mammals have been reported since 1976, when a group of dolphins died at an oceanarium in Hong Kong (Ocean Park). Melioidosis has been associated with the deaths of at least 49 cetaceans and 31 pinnipeds at Ocean Park over a period of 28 years.
At Ocean Park, diagnosis of melioidosis in marine mammals is confirmed by isolation, cultivation and biochemical identification of the organism. For suspected cases of melioidosis in cetaceans yielding a negative culture, diagnosis is supported by use of an antibody-capture enzyme-linked immunosorbent assay (ELISA) based on a protein-polysaccharide mixture of B. pseudomallei antigens. A cut-off is not defined for this ELISA and therefore its sensitivity and specificity have not been evaluated. A pinniped ELISA has not been previously developed for use at Ocean Park, and as a result, there is no diagnostic test available for pinnipeds suspected with melioidosis that yield a negative culture.
This study was conducted with the aim of developing and evaluating indirect antibody-capture ELISAs for serological diagnosis of melioidosis in cetacean and pinniped species kept at Ocean Park. The antigen used for coating the ELISA plates was B. pseudomallei exopolysaccharide. This antigen was previously used in an indirect ELISA developed for serological diagnosis of melioidosis in humans, achieving good sensitivity and specificity. Initially, isolates of B. pseudomallei cultured from a human patient and a dolphin were compared by biochemical characterisation, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot. After characterisation, B. pseudomallei exopolysaccharide was extracted by immunoaffinity chromatography, and various ELISAs were developed for serum testing.
For the cetaceans, two ELISAs--based on B. pseudomallei exopolysaccharide extracted by use of two monoclonal antibodies--were developed and their usefulness compared. Both cetacean ELISAs employed anti-dolphin IgG as secondary antibody. For each of the two groups of pinnipeds (phocids and otarids), four ELISAs were developed. Preliminary evaluation was carried out to compare the systems which employed the following secondary antibodies: 1) anti-grey seal IgG, 2) anti-Californian sea lion IgM, 3) anti-canine IgG, and 4) anti-canine IgM.
Based on the similar sensitivity and specificity values obtained, assays employing anti-canine IgG and IgM (which are commercially available and less costly) were used for further testing. A total of 199 serum samples collected from 32 cetaceans and a total of 200 serum samples collected from 35 pinnipeds were tested by the ELISAs. Five cetaceans and nine pinnipeds were culture-confirmed for melioidosis and four cetaceans and one pinniped were suspected as having melioidosis. Sequential serum samples were tested for both culture-confirmed and suspected cases. Samples from animals (outside of and within Ocean Park) without a prior history of melioidosis were also tested. The ELISAs were compared to the gold standard, culture and biochemical identification, for calculation of sensitivity and specificity. All ELISAs achieved high specificities, while sensitivities ranged for each test depending on time of sampling (number of days post-clinical presentation). In pinnipeds, IgG detection was a more effective diagnostic indicator of disease, when compared to IgM detection.