EFFECT OF FOUR DIFFERENT PRESERVATIVES ON AUTOFLUORENCE AND PEANUT AGGLUTIN LECTIN BINDING CHARACTERISTICS OF HAEMONCHUS EGGS.

Abstract

Haemonchus contortus is the most economically important trichostrongyle nematode infecting various ruminants. Fecal egg counts are used to measure the intensity of trichostrongyle infections, but egg morphology alone can't differentiate Haemonchus eggs from the other less pathogenic trichostrongyle genera also infecting ruminants. The lectin, peanut agglutinin (PNA), binds selectively to Haemonchus eggs and has become a useful tool for diagnosing Haemonchus infections during fecal egg counts. Unfortunately, this binding is not stable when eggs are stored either within fecal samples or when isolated from them; it completely disappears after 72 hr. Refrigeration or freezing doesn't improve its stability. The present study compared the PNA-binding intensities for Haemonchus eggs stored for 30 days in four different chemical preservatives with those of freshly isolated eggs. The four preservatives included neutral buffered formalin, sodium azide, povidone iodine, or 100% methanol. Rhodamine-conjugated PNA and a TRITC/rhodamine epifluorescence filter system were used for the study. Based upon PCR results, the egg sample used was primarily infected with H. contorus but also contained smaller amounts of Trichostrongylus (T.) axei. All of the eggs stored in each of the four preservatives were easily recognized as trichostrongyle eggs, exhibited some minor changes in morula size, and showed an increase in morula autofluorescence compared to unpreserved eggs. The extremely high autofluorescence for methanol-preserved eggs limits its applicability for any diagnostic protocols involving rhodamine and Haemonchus eggs. Autofluorescence intensities were similar among eggs stored in the other three preservatives. Specific PNA-rhodamine staining of Haemonchus eggs could be easily detected above the autofluorescence when neutral buffered formalin, sodium azide, or povidone iodine was used as the storage solution. This specific staining was highest for buffered formalin and lowest for povidone iodine. Haemonchus-positive eggs were defined as having PNA fluorescence intensity above their mean autofluorescence intensity (low specificity) or as having an intensity more than 20% higher than their autofluorescence (high specificity). The low and high specificity Haemonchus to Trichostrongylus ratios for eggs stored in buffered formalin and sodium azide were similar to those determined for unpreserved fresh eggs. This ratio was also similar for povidone iodine based on the low specificity criteria, but lower than the controls based on the high specificity criteria. These results suggest that neutral buffered formalin and sodium azide are the most appropriate preservatives based on the PNA staining results; however, other factors should also be considered when selecting a preservation solution for PNA-rhodamine studies involving Haemonchus eggs.