Characterization of hydatid cyst antigens by crossed immunoelectrophoresis and its implications for the serodiagnosis of hydatid disease in livestock

Phd - Thesis

Kydatidosis is a disease of major worldwide public health importance and with considerable economic consequences for the livestock industry. It shows a variable geographic distribution but reaches its highest levels in certain areas of Kenya and in South America. Meat inspection reports show that the disease is common in livestock in Kenya. A small survey carried out in this study showed a 14% prevalence of hydatid cysts in animals slaughtered in two abattoirs in Nairobi. In an attempt to find a specific antigen that can be used in an in vivo diagnostic test for livestock hydatidosis, it was considered appropriate to study the antigenic composition of hydatid cyst fluid (HCF), the stage of the parasite which is found in livestock. Hydatid cyst fluid was harvested from cysts which were collected from cattle, sheep and goats. A reference pool of concentrated HCF was made by mixing various preparations of HCF from these animals. In order to study the cross-reactivity of HCF antigens and other livestock parasites, saline homogenates of common parasites were also prepared. Polyvalent antisera against HCF and various parasites were raised in calves, sheep. goats and rabbits. Two monospecific antisera were raised against the two major HCF antigens A and B of Oriol ejt al.,(1971); (Am. J. Trop. Med. Hyg. 20, 569-574). All sera were absorbed with erythrocytes and polymerised cattle, sheep and goat plasma to remove any heterologous host contaminants as well as PI blood group substances. Double immunodiffusion tests were performed to search for antigens A and B in different hydatid cysts originating from different animal species and to study cross reactivity between HCF antigens and antigens of other parasites. Crossed immunoelectro- phoresis (CIEP) was used to characterize the HCF antigens and to study cross reactivity of these antigens and other parasites. The two major antigens in HCF (A and B) were precipitated out and jointly used in enzyme-linked immunosorbent assay (ELISA) to screen for hydatidosis in animals, A search for antigens A and B (which correspond to our antigens 5 and 4) revealed that sheep or goat liver cysts fluid was the best source of the two antigens. Fertile cysts were a superior source of the antigens than sterile cysts. Antigen 4 occurred more frequently than antigen 5. Sterile cattle lung cyst fluid was found to contain mostly antigen 4. Using CIEP, HCF was found to contain 13 antigens of parasite origin. These antigens were given numbers 1-13 according to their electrophoretic mobility, starting with the most anodic component. Although antigen 6 showed a faster mobility than antigen 5, the numbers were interchanged so that antign 5 would correspond to the "arc 5"'described by Capron and co-workers. All the antigens showed an anodic migration. Some antigens, especially 6 and 7, usually gave faint arcs and were not re'producible in every run. Antigens 4 and 5 gave the most prominent arcs and could easily be visualised before staining. In the double diffusion (DD) tests, HCF antigens were found to react with antigen extracts of Moniezia expansa, Cysticercus tenuicollis, Taenia saginata, Cysticercus bovis, Stilesia hepatica and Avitellina centripunctata. There were no reactions witli antigens of Haemonchus contortus, Spirocerca lupi, Oesophagostomum radiatum, Fasciola gigantica, Paramphistomum microbothrium, Ascaridia galli, Trichuris vulpis, Ascaris suum and Bunostomum phlebotomum. Using CIEP, all the 13 parasitic antigens, including Capron’s "arc 5" were found to cross-react with other parasites that were tested as mentioned above, Using antiserum specific for antigen 4 this antigen was detected in expansa, C, tenuicollis, T. saginata, C. bovis, S. hepatica, A, centripunctata, H. contortus, S. lupi and 0^_ radiatum. It was not found in gigantica, P, microbothrium, A. galli T. vulpis, A, suum and phlebotomum. Antigen 5 was found in expansa, C. bovis, C. tenuicollis, T. saginata and lupi but not in the other parasites. Crossed immunoelectrophoresis and DD methods were able to detect circulating antibodies in only a few animals naturally infected with hydatidosis. Antigens 4 and 5 were highly immunogenic in experimental immunization schedules. An attempt was made to utilise these two antigens in a recently established and highly sensitive test, ELISA. Based on the results of 180 samples, the sensitivity of the test was 981 while the specificity was 70%. In spite of the relatively low specificity, ELISA using the two antigens was considered a useful test in in vitro diagnosis of hydatidosis. In conclusion, it was found that HCF contained at least 13 antigens originating from the parasite, none of which was unique to the parasite. Antigens 4 and 5 were the main antigens present in HCF, The presence of the two antigens varied with the source of the cyst in that fertile sheep and goat hydatid cysts showed the most frequent occurrence of antigens 4 and 5 but sterile bovine lung hydatid cysts predominantly contained antigen 4. C vii) Cross reactivity with HCF antigens was common in species of cestodes and, to a lesser extent, nematodes and P. microbothrium. It may therefore be difficult to establish a serodiagnostic test for livestock hydatidosis because of the observed cross reactions. It would appear that the major obstacle to immunodiagnosis of livestock hydatidosis was metacestodes of T. saginata, Taenia hydatigena and Taenia ovis since the metacestodes are tissue parasites which tend to stimulate a high immunological response in the host

University of Nairobi