Development of diagnostic assays for sheep nematodes based on faecal antigen detection

Summary

​This report summarises a project designed to develop ELISA and dipstick assays to detect nematode infections in sheep by faecal analysis.  Such assays would have a significant effect on the sheep industry by allowing graziers to know the level and types of worms infecting their livestock.  This information would allow decisions on the timing of drug treatments and which sheep need treatment to mitigate the effects of infection.  In addition, graziers would be able to use the tests to determine which of their stud sheep can maintain low parasite burdens and are suitable for breeding parasite resistant offspring.  
The methods used in these studies included specific antigen isolation from infected sheep faeces and from adult worm excretions and secretions (ES) collected under in vitro culture conditions.  Antibodies were raised in rabbits, chickens, mice and guinea pigs and then tested in a range of immunological assays to determine their antigen specificity in faecal preparations and ES products, their sensitivity and specificity in ELISA and ability to discriminate infected from uninfected faeces in dipstick assays.  Problems occurred mainly as a consequence of the close relationship between these parasites and thus the sharing of many protein antigens and in the contamination of adult nematode ES with sheep proteins.  These problems confounded the attempt to produce specific assays for each species and caused non-specific reactions especially when testing faecal material. A specific Haemonchus antigen was isolated from faeces and adult ES and this was used to produce antibodies which together with a substrate binding reaction allowed the development of a specific Haemonchus ELISA.  A dipstick was then developed to differentiate Haemonchus infected faecal solutions using a sandwich double-antibody, binding assay.  
The finding of sheep protein contamination in Osteratgia ES led to the development of new methods for the isolation of adult nematode antigens.  Antibodies raised against the new ES were more specific though still reacted with all 3 species of nematode.  This allowed the development of an ELISA and dipstick assay that could detect all three nematode species in faecal solutions.  Work on Trichostrongylus resulted in a similar conclusion and thus in the final analysis two tests were developed during the project.  The first detects Haemonchus infections and the second detects all three nematode infections.  If combined on one dipstick they would allow a grazier to determine 

1. whether his sheep have worms and 

2. if those infections include a significant number of Haemonchus worms. 

Such information is a significant improvement on current faecal diagnostic methods and as the tests should be able to be carried out with very little training and next to the drenching race; this work has the potential to significantly change current strategies for control of nematode infections