Immunochemistry of Fasciola hepatica in the rat model

The economic impact of fasciolosis on livestock industries including those in tropical countries such as Nigeria has prompted research into various aspects of the host-parasite relationship in fasciolosis. An improved knowledge of this interaction should contribute to the development of control measures for fasciolosis. The excretions, secretions and surface components of a parasite are by their nature centrally involved in the host/parasite interaction. Rats, like cattle, are capable of developing resistance to fasciolosis after primary infection and are therefore considered a suitable laboratory model for cattle. The objective of this study was to characterise the excretory/secretory (ES) and surface components of Fasciola hepatica as it develops in the rat, and to identify those components involved in the host/parasite interaction that may have diagnostic and/or protective value.

In the first instance the available scientific literature on the immunochemical nature of Fasciola spp. and on natural and experimentally induced resistance to F. hepatica infection in various hosts was reviewed.

The rat sera from the three trials were checked for the presence of protective antibodies by passive protection studies. Only in the latter two trials was the level of protection conferred on recipient statistically significant. The probable causes for the lack of significant protection in trial 1 are discussed.

The silver stained protein profiles of ES from newly excysted (DO) flukes and one-day old (Dl) flukes were characteristic and were similar to each other. The ES of parenchymal 14-day old (D14) and adult (D56) flukes were markedly different from DO and Dl flukes but similar to each other. The silver stained total xiv ES protein profiles of the developing flukes were very different from the total biosynthetically (35S-methionine) radio-labelled ES protein profiles. Possible reasons for this are discussed. However, as with the total silver stained ES protein profiles there were clear changes in the profiles of the biosynthetically radiolabelled ES as the flukes developed. It is suggested that these differences in the ES products may reflect the changing environment and activities of the flukes. The possible functions of the changing ES products are discussed.

Western blot studies of adult fluke ES using sera collected sequentially from rats infected with F. hepatica in the three trials revealed a 25 kDa doublet (reduced) and 26 and 27 kDa antigens (non-reduced). It is thought possible that these antigens provoke a protective antibody response. The rat sera (I2) used for the second protection trial contained more antibodies which reacted with these particular antigens than the sera used in the other 2 trials and had a demonstrably greater protective ability. The 25 kDa doublet was also identified in D14 ES but not in the DO or D1 ES. In DO and D1 ES, however, an immunodominant reduction-sensitive 191kDa antigen was identified.

Western blot studies on DO, D14 and adult ES using sequential bleeds from rats injected with the protective I2 serum prior to challenge infection revealed two interesting points. Firstly, in rats that were completely protected from infection antibody activity against the 25, 26 and 27 kDa antigens in D14 and adult fluke ES and against the 191 kDa antigen in DO and D1 ES gradually declined as the experiment progressed. Secondly, in partially protected rats although the response to these antigens had also declined by D14 and D28, it then rose again by D42 and D56. Thus, although these antigens may be involved in promoting a protective immune response, the antibodies against them are also good indicators of infection.

Immune-coprecipitation of biosynthetically radio-labelled DO ES with sera from infected rats in trial 2 revealed a 26 kDa antigen. It is suggested that this antigen may be the same as the 25 kDa antigen detected in western blots of D14 and adult fluke ES and that it was not detected in Western blots of DO and D1 ES due to the limited assay sensitivity. A novel antigen of about 38kDa was strongly detected by serum I2 containing protective antibody in immune-coprecipitation of biosynthetically radio-labelled ES of parenchymal (D14, D28 and D42) flukes. It is suggested that this antigen may also have protective and/or diagnostic properties.

The study thus indicated that the following F. hepatica antigens merit further study; the 191kDa antigen in DO ES, the 25 kDa in D14 ES and the 25, 26 and 27 kDa antigens in adult ES, in order to further elucidate their role in the host/parasite relationship. Similarly, the 26 kDa antigen detected in immunocoprecipitation of biosynthetically (35S-methionine) radio-labelled DO ES, and the 38 kDa antigen detected in D14, D28 and D42 ES deserve further investigation. The fact that these antigens were being actively synthesized by the parasite at the time of study suggests that it would be feasible to clone them.

In conclusion, the possible relevance of the above antigens for diagnosis and protection in fasciolosis is discussed, as is the potential use of non-immunogenic biosynthetically radio-labelled ES components in diagnostic assays designed to detect parasite products. Some components of this nature were detected in the course of this study.

Overall, the study has clearly identified antigenic components in the ES and surface extracts of F. hepatica which are involved in the host/parasite interaction in the rat model and, by implication, may also be of significance in bovine fasciolosis. The antigens detected by western blotting and by immuno-coprecipitation of DO ES (that is those of 191 and 26 kDa) as well as the 38 kDa antigen detected in the ES from parenchymal flukes have not been previously reported in the rat model.

Three trials were conducted during the study in order to produce supplies of rat antiserum which was protective against F. hepatica. Rats were infected with either 10 (first trial) or 20 (second and third trials) F. hepatica metacercariae as information from the literature indicated that these doses were adequate to stimulate the production of protective antibody levels.