Immunodiagnosis of Bovine Cysticercosis using selected antigens

PhD Thesis

Despite significant advances in the development of cestocidal agents and improved understanding of the epidemiological factors affecting the transmission of tapeworms, there has been in fact an increase in human taeniasis and bovine cysticercosis due to Taenia saginata during the last two decades. The control measures whicl) have been tried have largely failed to achieve the expected results. In theory, this cyclozoonosis can easily be controlled. In order to break the cycle of man-cattie-man transmission, two approaches, or a combination of the two, are evident. Firstly, the eradication of human taeniasiz through appropriate treatment, thus eliminating the source of infection for cattle. This approach has largely failed because of the logistics involved, in spite of the availability of efficient and non-toxic drugs. The second logical approach would be to prevent infected meat from r£aching the market. This approach is presently in operation in the form of meat inspection legislation and procedures, which inter alia are aimed at the detection of ~ saginata metacestodes to prevent marketing of infective beef. Unfor.tunately. experience and careful studies have shown that the meat inspection procedures are unable to detect light infection, resulting in the marketing of considerable quantities of infective beef. In view of the present unsatisfactory results of attempts to control human taeniasis and bovine cysticercosis, it could be surmised -that a serological diagnostic method might be of value both in the ante-mortem as well as post-mortem diagnosis of bovine cysticercosis. It is envisaged that a reliable ante-mortem diagnostic method would provide the livestock farmer with means of identifying infected animals. Such animals may be disposed of immediately to prevent the losses incurred in feeding of animals which may be condemned or downgraded at the time of slaughter. A serological test may also assist in identifying infected animals at the time of slaughter, despite of negative findings during meat inspection. In the future, cattle herds could be screened using a serological method. Those giving positive reactions may be considered for treatment when an efficient drug becomes available. So far, however, drug treatment of bovine cysticercosis has not been attempted except on an experimental basis. Finally, it can be envisaged that the present meat inspection procedure can be simplified through the introduction of a serological method for bovine cysticercosis, in a way similar to what has been done for porcine trichinellosis. The fundamental aim of the present study was to examine the antigenic components of T.saginata metacestodes in an attempt to select antigen(s) suitable for the use in serological tests. This required investigations of the complexity of antigenic determinants shared with other common paLasites of cattle, the selection of antigen(s) possessing adequate specificity and the establishment of sensitive seLological methods using these antigens. In addition to the potential pLactical implications of the development of such irr~unonssnys, these investigations might contLibute to the undeLstanding of funda~ental asp~cts of the inunun~r-esponae s to par-as i t i,c infections in gener-a l. Chaptet' I describes the establishment of a reference system for the antigens of 1saginata metacestodes. Fifteen antigens wet'e defined by the Laurell crossed immunoelectrophoresis technique. Antigens were given abitLary numbers starting with the most anodic components. Antigens 1,3,4,5,7,8,9,10,11, and 13 were found to be shaLed betwecn T. saginata cysts and the adult wor"" while Antigens 1,2,4,6,7,9,10,12 and 13 were common among cestodes. Antigens 10,14 and 15 were shared with con~only occurLing nematodes and trematodes of domestic animals. Antigens 4, 8 and 11 were considered potentially useful and were selected for furthet' study. Antigen 4 was found to be common among all cestodes examined, while Antigens 8 and 11 had a more restricted di st rIbuti.on . Antigen 8 was only shared \-lith 1. saginata and its metacestode. Specific antisera to these antigens wer-e pr-epar-ed in rabbi t s using iImnunoprecipitates obtained in i~~unodiffusion tests using rabbit antiserum to 1saginata metacestodes, and metacestode extrect as untigen. Chapter II describes the isolation and purification procedures of these antigens. All the three antigens were isolated using an affinity chromatography technique. The antigens were analysed by isoelectric focusing and polyacrylamide gel electropporesis in which sodium dodecyl sulphate ~as incorporated (SDS-PAGE). Antigens 4,8 and 11 were found to have molecular weights of 63 kd, 260 kd, and 68 kd and save pI values of 5.20, 6.95 and 5.50, respectively. Antigen 8 was found to consist of three components of molecular weights 110 kd, 72 kd and 61 kd. All these components showed reactions of identity in.immunodiffusion tests, indicating that they shared the same epitopes. Chapter III describes an attempt to determine whether the three antigens could be used for the serodiagnosis of bovine cysticercosis. The "sandwich"" enzymeimmunoassay and a radioimmunoassay were first used to evaluate these antigens using sera from experimentally infected animals. since antibodies to all three antigens could be detected in experimentally infected animals, the applicability of these antigens in detecting antibodies in naturally infected animals was evaluated. The "sandwich" enzyme immunoassay using Antigen 8 gave a sensitivity of 66~, a specificity of 40~and a predictive value of 56~. Antigen 11 gave a sensitivity of 55~, a specificity of 48~, and a predictive value of 52~. Antigen 4 gave a sensitivity of 45~, a specificity of 45~ and a predictive value of 46~. (vii) There was no statistically significant difference between the sensitivities obtained with Antigen 8 and 11 in the enzyme irrmunoassay (t = 1.925t P> 0.05). There was a significant difference in the sensitivities obtained when Antigen 8 was compared with Antigen 4 (t = 2.528~ P<0.05) Antigen 8 was more sensitive than f~tigen 4. A comparison between Antigen 11 and Antigen 4 also gave a significant difference in sensitivity (t = 5.885~ ~0.05) with Antigen 11 giving a higher sensitivity than Antigen 4. There was no statistical difference between the specificities obtained when Antigen 8 was compared with Antigen 11 in enzymeiwmunoassay (t = 0.980; P)0.05), while there was a statistically significant difference in specificities shown by Antigen 8 and Antigen 4 (t 3.261; P< 0.05), with Antigen 8 giving a higher specificity than Antigen 4. Antigen 11 gave a higher specificily than Antigen 4 (t = 2.297; P<"0.05). In the radioimmunoassay, using labelled antigens, a sensitivity of 82%, a specificicity of 25% and a predictive value of 58% were obtained with Antigen 8. A sensitivity value of 76%, a specificity of 30% and a predictive value of 58% were obtained with Antigen 11. Antigen 4 was not evaluated in this assay. There was no statistical difference when the sensitivities obtained with P~tigen 8 were compared with Antigen 11 (t = 0.712: P> 0.05). Neither was there any difference in the specificities obtained with these antigens in radioimmunoassays (t = 1.05/,; P.) 0.05) . There was a statistical difference in sensitivity when Antigen 8 was used in radioinununoassay, as compared to its use in enzymeirr~unoassay (t = 3.803~ P~.05) with radioimmunoassays using Antigen 8 giving a higher sensitivity. There was also a difference in specificities obtained in radioimmunoassay as compared to enzymeimmunoassay (t = 2.042; P <.0.05) with enzymeinununoassay using Antigen 8 giving a higher specificity. A significant difference in sensitivity was obtained when Antigen 11 was used in a radioimmunoassay as compared to enzymeimmunoassay (t = 2.617; P< 0.05) with enzyme i~~unoassay using Antigen 11 being more specific. This difference in sensitivities and specificities could be attributed to the use in the radioimmunoassay of labelled antigens, while in the "sandwich" enzyme immunoassay an anti-bovine I~G conjugate was used. The high percentage of apparent false positive reactions obtained with the immunoassays may in fact be true positive reactions because of the poor reliability of meat inspection procedures which were used as the definitive parameter for the diagnosis. On the other hand, false positive reactions may have been due to abortive infections as the results of the ingestion of senescent eggs. Such eggs may fail to develop into matur.e cysts but nevertheless tr.igger an irr~uneresponse and give rise to detectable antibody levels. There are severaI ways in which the i.mmunoassays describ8d in this study may be applied. For example, the enzymeimmunoassay using Antigen 8 and 11 may be used for the selection of animals which are heavily infected. Such animals could be separated for treatment or slaughtered at an early age, thereby saving the farmer from keeping animals which can be downgraded or condemned at the time of slaughter. The same method could be.used in the selection of animals which are to be taken to a feedlot for fattening. These assays could also be used for the detection of lightly infected animals as an adjunct to the current meat inspection procedure. This could justify the cold storage of all carcasses from animals which gave a positive reaction in the immunoassay. On the other hand, animals \ihich gave negative reactions in the serological tests could be subjected to a less vigorous inspection. The presence of antibodies to T. saginata metacestodes in cattle from a certain farm or area may also be used as indirect way of identifying persons with taenia£is. The treatment of persons identified in this way should be an intergral part of control measures for bovine cysticercosis. In this study it has been sho~m that the selection of certain antigens of T. saginata metacestodes can easily be made from complex mixture of antigens by the use of a reference pattern based on the Laurell crossed immunoelectrophoresis technique. The same r-ef er-enc e sys tem can be used to elucidate the relationship of these antigens with those of other parasites. The selection of three antigens which were consistently present in T. saginata metacestodes and absent in most other prasites was considered the most appropriate choice of antigens for use in specific serodiagnostic tests for bovine cysticercosis. Encoura~ing results were obtained with Antigens 8 and 11 and these antigens could be used as an adjunct to current meat inspection procedures.