Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes.

BACKGROUND: The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. RESULTS: 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45-11.5 ng/µL, and on Qubit™ ranged between undetectable - 0.962 ng/µL. Median A260/280 ranged between 0.505-3.925, and median A260/230 ranged - 0.005 - 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. CONCLUSIONS: A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

A pilot study to investigate the measurement of immunoglobulin A in Welsh Cob and Welsh Pony foals' faeces and their dam's milk.

Aims: To determine if an ELISA for measurement of IgA in equine serum could be used to measure concentrations of IgA in foal faeces and to determine correlations with concentrations in the milk of the dam.Methods: Faeces from 20 Welsh Cob and Welsh Pony foals and milk from their dams were collected within 12 hours (Day 0) and at 6 days after parturition (Day 6). On Day 6, faeces could not be collected from 2/20 foals, and milk samples could not be collected from 3/20 mares. An equine IgA ELISA validated for serum and plasma was used to measure concentrations of IgA in all samples in triplicate. The precision of the assay for each sample type was determined using modified CV.Results: IgA was not detectable in 7/20 Day 0 faecal samples and in 2/18 Day 6 faecal samples. For samples with detectable IgA, the mean modified CV was 10.5 (95% CI = 6.0-15.0)% for Day 0 faecal samples, and was 6.8 (95% CI = 4.3-9.4)% for Day 6 faecal samples. Median concentrations of IgA in faeces on Day 0 were lower than concentrations on Day 6 (0.7 mg/g vs. 37 mg/g dry matter; p = 0.003). Concentrations of IgA in milk and faeces on Day 6 were statistically correlated (r = 0.59; p = 0.006).Conclusions and clinical relevance: The IgA ELISA showed acceptable precision when used to estimate concentrations of IgA in foal faeces during the first week of life, but IgA could not be detected in 37% of meconium samples collected on Day 0. This assay may be useful for investigation of the role of maternal milk IgA in the gastrointestinal tract of neonatal foals, but further assessment of both accuracy and precision of the ELISA is required.

Bioinformatic analysis of eosinophil activity and its implications for model and target species.

Eosinophils are important immune cells that have been implicated in resistance to gastrointestinal nematode (GIN) infections in both naturally and experimentally infected sheep. Proteins of particular importance appear to be IgA-Fc alpha receptor (FcαRI), C-C chemokine receptor type 3 (CCR3), proteoglycan 3 (PRG3, major basic protein 2) and EPX (eosinophil peroxidase). We used known human nucleotide sequences to search the ruminant genomes, followed by translation to protein and sequence alignments to visualize differences between sequences and species. Where a sequence was retrieved for cow, but not for sheep and goat, this was used additionally as a reference sequence. In this review, we show that eosinophil function varies among host species. Consequently, investigations into the mechanisms of ruminant immune responses to GIN should be conducted using the natural host. Specifically, we address differences in protein sequence and structure for eosinophil proteins.