Egg autofluorescence and options for detecting peanut agglutinin binding for the identification of Haemonchus contortus eggs in fecal samples.

Quantifying eggs from Haemonchus and other trichostrongyle genera in sheep and goat fecal samples is important for evaluating control and treatment strategies for this family of nematodes with divergent pathologies, capabilities for anthelmintic resistance and environmental susceptibilities. Unfortunately, egg morphology among most of the genera do not differ enough to support the accurate identification of these genera with standard microscopic techniques. Several studies have identified specific lectins which bind selectively to sugars located on the egg surfaces for individual genera among the trichostrongyles. To detect lectins binding to these eggs, they must be directly or indirectly bound to fluorophores, and observed with an epi-fluorescence microscope. The binding of multiple lectins to isolated eggs from a fecal sample can be simultaneously detected if fluorophores are used whose excitation and emission spectra do not overlap, and this would enable the development of a fluorescence-based diagnostic test that identifies multiple trichostrongyle genera within each sample. The present study compared the usefulness of different, commercially available detection systems for use in detecting lectin binding to trichostrongyle eggs. Comparisons were made using the detection of PNA binding to H. contortus eggs with the goal of finding three systems with color spectra that do not overlap. These evaluations included both fluorophores directly conjugated to PNA in a one-step incubation protocol and a two-step incubation protocol involving biotinylated PNA and streptavidin conjugated to different fluorophores. Autofluorescence can affect the efficiency of any fluorescence-based detection system, and significant autofluorescence was observed among the unstained H. contortus eggs with the DAPI-type fluorescence filter, but it was significantly lower with the FITC-type filter and was virtually absent with the rhodamine-type filter. This study demonstrated that all the PNA detection methods tested with H. contortus eggs generated fluorescence intensities (FIs) that were significantly above the autofluorescence generated by the eggs among the three different fluorescence filters. Fluorescence intensities from PNA directly conjugated to either the FITC or rhodamine fluorophores were not different, but the lower autofluoresence in the rhodamine-type filter will enable this fluorophore to be detected more efficiently. Use of biotinylated PNA combined with streptavidin-conjugated to synthetic fluorophores (Alexa Fluor 405, 488 and 546) significantly increased FIs over that of the directly conjugated PNA, but there were no significant differences in FIs among these three biotin-avidin conjugation fluorophores. This biotin-avidin system required two incubation steps. Doubling the concentration of PNA also provided increased FI, at least for the biotin-avidin system. Adding an additional amplification step to the biotin-avidin system involving biotinylated anti-streptavidin followed by the streptavidin-Alexa Fluor complex also provided additional fluorescence.