Development of a Reverse Passive Latex Agglutination Test for Swine Dysentery

Veterinary science, Animal diseases

Thom, Fiona

University:University of Glasgow

关键词: Veterinary science, Animal diseases;   

Others  :  http://theses.gla.ac.uk/74725/1/11007644.pdf

来源: University of Glasgow

【 摘 要 】

Two diagnostic reverse passive latex agglutination tests for swine dysentery were produced and evaluated. Such tests are faster and potentially more specific than current diagnostic tests. In the first test the antibody used was raised in a rabbit to whole Serpulina hyodysenteriae, S80/5 cells. The antiserum was absorbed with non-S. hyodysenteriae spirochaetes PWS/A and 4/71 and used to coat 0.46mumlatex beads. These beads were suspended in glycine buffered saline pH 8.2. This reagent was found to detect S. hyodysenteriae at 10e8 organisms/ml and the type strain B78 at 10e8 organisms/ml. The test also cross reacted with PWS/A and 4/71 at levels of 10e9 organisms/ml and 10e11 organisms/ml respectively. When cultured S. hyodysenteriae was suspended in normal pig faeces, levels of 10e8 organisms/ml could be detected. When faeces from experimentally infected pigs was examined, RPLA positive faeces was found to contain 6 x 10e8 organisms/ml. In the first instance, the test specificity was examined for its ability to distinguish between S. hyodysenteriae and other intestinal porcine spirochaetes and the sensitivity was looked by determining the lowest concentration of organisms detectable by the test. However when dealing with diagnostic tests, the sensitivity of the test should also be looked at as the number of positive results from animals with the disease. The specificity as the proportion of animals without the disease which have a negative result. This was looked at using field samples from pigs with suspected swine dysentery. Twenty eight field samples of dysentery or diarrhoea were examined by the RPLA and culture. Twenty three were positive by RPLA but only one yielded S. hyodysenteriae Eleven others yielded spirochaetes shown by the haemolysis pattern and API ZYM not to be S. hyodysenteriae. The remaining eleven samples contained spirochaetes visible in smears but which could not be grown. In an attempt to improve specificity antiserum was prepared in a rabbit to the 16 kDa antigen of S. hyodysenteriae purified by SDS PAGE and immunoblotting. The resulting polyclonal antiserum was used to prepare a second test. This was more sensitive detecting S.hyodysenteriae at levels of 10e6 organisms/ml and non-S. hyodysenteriae (PWS/A and 4/71) at 10e9-10e10 organisms/ml. It was used on the samples previously tested with the addition of 17 further fresh samples. The test appeared to be more sensitive and more specific than before, but false positives still occurred. Examination by SDS PAGE of 14 of the non-S. hyodysenteriae isolates revealed that they contained a 16 kDa antigen. Negative staining of 1 of these spirochaetes confirmed that their ultrastructure differed from that of S. hyodysenteriae. It appeared that the false positives might result from the possession of a 16 kDa antigen by non-S. hyodysenteriae spirochaetes.