Development and evaluation of an improved assay for detecting specific antibodies post contagious caprine pleuropneumonia exposure

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Wambugu, Anderson Njagi
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The diagnosis of contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subspecies capripneumoniae relies on the isolation and identification of the organism as well as different serological tests. The various serological tests include Complement Fixation Test (CFT), Indirect Enzyme- Linked Immunosorbent Assay (I-ELISA) and a field Latex Agglutination test (LAT). However, these tests have a number of drawbacks including low specificity and sensitivity. Further, the in-vitro culture and isolation of the mycoplasma requires highly skilled laboratory personnel. This study was designed to develop an I-ELISA using a unique and specific capsular polysaccharide epitope to Mycoplasma capricolum subspecies capripneumoniae in order to improve the diagnosis of CCPP relative to that of the field LAT based on the same unique and specific epitope. One precharacterized Mycoplasma isolate for CCPP and a control isolate from contagious bovine pleuropneumonia (CBPP) were cultured to log phase and spun in a refrigerated centrifuge to harvest separately cells and supernatant. The polysaccharides were extracted from the supernatants and used for sensitising latex beads and coating of ELISA plates after initial optimisation for the assays. Both tests used the extracted and standardised CCPP specific polysaccharide as the antigen to detect homologous antibody. LAT was performed on undiluted sera whereas I-ELISA was performed on two different serum dilutions of each serum sample tested. The diagnostic sensitivity of the developed I-ELISA and LAT was estimated using 148 sera from CCPP infected goats from a farm in Kima and the diagnostic specificity of both the IELISA and LAT was estimated from 344 CCPP uninfected goat sera obtained from Kabete, Kajiado and Somalia. Positive and negative predictive values and the likehood ratios were estimated using the calculated diagnostic sensitivity and specificity. Both tests had high analytical specificity while I-ELISA had a higher analytical sensitivity than LAT. However, both test had low diagnostic sensitivity and high diagnostic specificity. Predictive values and calculated likehood ratios suggested that I-ELISA at 1:500 sera dilution was more likely to correctly classify truly exposed goats and that LAT was more likely to correctly classify truly unexposed goats. In conclusion, although diagnostic sensitivity of the developed I-ELISA needs to be improved, the high analytical and diagnostic specificity, objectivity and its capacity to be adapted to test large number of sera in short periods of time are advantages not provided by LAT. The use of this I-ELISA will facilitate epidemiological studies on CCPP infections. This is especially important in developing countries where areas of enzootic instability to CCPP infections are not well defined, precluding the application of preventive measures with economical rationality.
Department of Biochemistry and Biotechnology, 102p. The SF 969.P8W3 2005
Pulmonary adenomatosis, Goats--Disease, Pleuropneumonia