Triple lectin staining of trichostrongyle eggs from naturally infected small ruminants

Abstract

Trichostrongyle nematodes can be a major threat to the profitability of small ruminant producers depending of the species and intensity of trichostrongyles parasitizing their herd. Haemonchus contortus, Teladorsagia circumcincta, and Trichostrongylus colubriformis are typically the most common and clinically important species. Three lectins (PNA, LCA and AAL) have been reported to bind specifically to eggs from these three genera and therefore could be used to quantify the intensity of each species in individual animals. Peanut agglutinin (PNA) has been the most commonly tested lectin because it selectively binds intensely to eggs of the most pathogenic species, H. contortus. Lens culinaris agglutinin (LCA) and Aleuria aurantia agglutinin (AAL) have shown specificity to T. circumcincta and Trichostrongylus spp. respectively, however, these lectins have only been evaluated using eggs harvested directly from adult females, and not from fecal samples. The purpose of the present study is to describe a method to sequentially stain H. contortus, T. circumcincta and Trichostrongylus spp. fecal eggs with PNA, LCA and AAL, and then evaluate the resultant staining patterns seen with eggs collected from a naturally infected goat shown with PCR to contain H. contortus, T. circumcincta, Ostertagia leptospicularis, Trichostrongylus colubriformis and Trichostrongylus axei eggs. These results were also compared with patterns observed with eggs stained with single lectins and double combinations of lectins. The various patterns were then compared to those seen with egg samples collected from an ewe shown to only contain H. contortus. PNA bound intensely and uniformly to all eggs from samples containing only H. contortus eggs; however, some eggs additionally bound LCA and AAL in localized patches of varying size, and a few eggs exhibited intense and uniform binding of all three lectins. Single PNA-staining of goat samples containing the five trichostrongyles species identified most eggs as H. contortus, and triple-staining showed patterns consistent with those seen for H. contortus. Binding of AAL to non-Haemonchus eggs was uniform but showed significant variations in intensity. Lesser staining eggs tended to also stain intensely with LCA, which is consistent with published binding pattern for T. circumcincta. Most eggs that AAL bound intensely to did not bind with LCA, which is consistent with published binding pattern for Trichostrongylus spp. Autofluorescence was observed with the DAPI filter-cube among most non-Haemonchus eggs. This study demonstrates the need for additional field studies to further validate the specificity of these three lectins for use in identifying eggs from the three species of trichostrongyles.

Keywords: Aleuria aurantia agglutinin (AAL); Diagnosis; Fecal eggs; Goats; Haemonchus contortus; Lectin; Lens culinaris agglutinin (LCA); Peanut agglutinin (PNA); Sheep; Teladorsagia circumcincta; Trichostrongylus.