Comparison of three different preservatives for morphological and real-time PCR analyses of Haemonchus contortus eggs.

Despite the development of several recent PCR assays for the egg stages of various trichostrongyles, there have been no protocols described for preserving field samples for PCR without refrigeration. In this study, Lugol's iodine (LI), sodium azide (SA), and neutral buffered formalin (NBF) were evaluated using Haemonchus contortus eggs to determine their potential as a preservative for trichostrongyle egg samples to be processed with real-time PCR. When egg recovery, embryo development, and egg morphology were evaluated from fecal samples preserved with LI, NBF, and SA, there was equally good recovery and preservation for the first month. Preserved eggs were detectable for 1 month, but after 6 months, none could be recovered. When real-time PCR analysis was performed on eggs isolated from faeces preserved with LI and SA, there was no detectable inhibition compared to fresh, non-preserved eggs; however, NBF significantly inhibited amplification. The results from this study demonstrate that for PCR applications LI and SA are effective preservatives for H. contortus eggs, resulting in good preservation of morphology while allowing for uninhibited PCR.

Real-time PCR for quantifying Haemonchus contortus eggs and potential limiting factors.

The purpose of this study was to evaluate the practicality of using real-time PCR for quantifying feces-derived trichostrongyle eggs. Haemonchus contortus eggs were used to evaluate fecal contaminants, time after egg embryonation, and the presence of competing and non-competing DNAs as factors that might interfere with generating reproducible results during simplex and multiplex quantitative real-time PCR (QPCR). Real-time PCR results showed linear quantifiable amplification with DNA from five to 75 eggs. However, threshold cycle (C (T)) values obtained by amplification of DNA from egg numbers between 75 and 1,000 did not differ significantly. Inhibitors of QPCR were effectively removed during DNA extraction as exemplified by the absence of any improvement in C (T) values with bovine serum albumin or phytase treatments. Changes from egg embryonation could only be detected during the first 6 h. Noncompetitive DNA did not appear to impact amplification; however, in a multiplex reaction a competing trichostrongyle such as Cooperia oncophora can hinder amplification of H. contortus DNA, when present at tenfold greater amounts. This study demonstrates the usefulness of QPCR for amplification and quantification of trichostrongyle eggs, and identifies potential limitations, which may be addressed through multiplex assays or the addition of a standard: exogenous DNA target.