Modification of two PCR-based assays for use in differentiating trichostrongyle eggs and their validation with naturally-infected calf fecal samples.

摘要

Trichostrongyle nematodes parasitize ruminants throughout the world causing disease and lost productivity. Understanding the epidemiology of these nematodes is crucial to improving control strategies which reduce these losses. Previous research has focused on developing PCR-based assays for genus-level differentiation, however, little progress has been made in preparing these assays for field use. The focus of this dissertation is to prepare and validate two assays, a traditional multiplex PCR (MPCR) and quantitative real-time PCR (QPCR) assay, for field studies.The first step in preparing these assays for use with "field samples" was to establish an effective eggshell disruption and DNA isolation protocol for trichostrongyle eggs. Purified Ostertagia ostertagi eggs were found to be best disrupted with a bead beater utilizing ceramic beads. The DNeasy Plant kit (Qiagen) was found to be optimal.To determine the potential for real-time PCR quantification, isolated groups of H. contortus eggs were amplified and assessed for correlation of egg numbers and CT values. Results indicate that from 5 to 75 eggs, amplification is linear (R2=0.98), and C T values for each egg quantity are significantly different. When potential limiting factors were assessed, larval development and competitive effects (in a multiplex reaction) were shown to inhibit quantification.In preparation for future field studies, a suitable trichostrongyle egg preservative for PCR analysis needed to be identified. Lugol's iodine (LI, a known nematocide), sodium azide (SA), and formalin (NBF) were evaluated with H. contortus eggs. Egg morphology was preserved with uninhibited real-time PCR with both SA and LI preserved feces. Inhibition was seen with NBF in all samples.When field validation was carried out with both PCR assays, high sensitivity (>90% for Cooperia and Haemonchus, and 79% for Ostertagia spp.) was seen when comparisons were made to larval culture data. Additional samples from 13 herds showed high sensitivity, near 80%, for both assays, similar to recent studies with human hookworms. The results from this research indicated that both QPCR and MPCR can be utilized in field studies by combining the egg DNA isolation protocol. Preservation can be performed, and if eggs are isolated, quantification may also be possible.