A new enabling proteomics methodology to investigate membrane associated proteins from parasitic nematodes: case study using ivermectin resistant and ivermectin susceptible isolates of Caenorhabditis elegans and Haemonchus contortus.

The mechanisms involved in anthelmintic resistance (AR) are complex but a greater understanding of AR management is essential for effective and sustainable control of parasitic helminth worms in livestock. Current tests to measure AR are time consuming and can be technically problematic, gold standard diagnostics are therefore urgently required to assist in combatting the threat from drug resistant parasites. For anthelmintics such as ivermectin (IVM), target proteins may be present in the cellular membrane. As proteins usually act in complexes and not in isolation, AR may develop and be measurable in the target associated proteins present in the parasite membrane. The model nematode Caenorhabditis elegans was used to develop a sub-proteomic assay to measure protein expression differences, between IVM resistant and IVM susceptible isolates in the presence and absence of drug challenge. Evaluation of detergents including CHAPS, ASB-14, C7BzO, Triton ×100 and TBP (tributyl phosphine) determined optimal conditions for the resolution of membrane proteins in Two Dimensional Gel Electrophoresis (2DE). These sub-proteomic methodologies were then translated and evaluated using IVM-susceptible and IVM-resistant Haemonchus contortus; a pathogenic blood feeding parasitic nematode which is of global importance in livestock health, welfare and productivity. We have demonstrated the successful resolution of membrane associated proteins from both C. elegans and H. contortus isolates, using a combination of CHAPS and the zwitterionic amphiphilic surfactant ASB-14 to further support the detection of markers for AR.

The soluble proteome phenotypes of ivermectin resistant and ivermectin susceptible Haemonchus contortus females compared.

Anthelmintics in the absence of vaccines have underpinned a parasite control strategy for over 50 years. However, the continued development of anthelmintic resistance (AR) threatens this control. Measuring early AR is difficult as there many routes that resistance can arise from within multi-nematode populations operating complex metabolism capabilities coupled to different drug management pressures. There is an urgent need to identify and measure early resistance in the field situation. Proteomic profiling of expressed soluble proteins offers a new approach to reveal a drug resistant phenotype within a complex protein pattern. The hypothesis under test was that established differences in drug response phenotypes between nematode isolates can also be measured in their comparative proteomes. As a case study, proteomic differences were measured between an ivermectin resistant and susceptible adult female Haemonchus contortus. Adult H. contortus females were extracted from the abomasa of six lambs. The nematodes had been maintained in the lambs as monospecific isolates of either ivermectin susceptible or ivermectin resistant worms. Comparative analysis of the soluble proteome was completed along with immuno-proteomic analysis using pooled infection sera from the lambs. Following image analysis, spots of interest were excised and analysed by peptide mass fingerprinting and the proteins putatively identified using BLAST. Overall, a relative increase in the expression of proteins involved in the detoxification metabolic area was observed in the resistant isolate. In addition, Western blotting analysis also revealed differences in immuno-reactivity profiles between resistant and susceptible isolates. It can be concluded from this study that proteomic differences can be detectable between ivermectin susceptible and a resistant isolates of H. contortus, which could be further explored using other isolates to confirm if proteomic based fingerprinting offers molecular phenotyping or a new panel of resistance biomarkers.

Effect of the level of iodine in the diet of pregnant ewes on the concentration of immunoglobulin G in the plasma of neonatal lambs following the consumption of colostrum.

Excessive I in the diet of pregnant sheep can reduce the concentration of antibodies in the blood plasma of the lambs after they have consumed colostrum. Our aim was to determine the dose of dietary I that would avoid this effect, and to relate this to changes in the concentrations of hormones and metabolites in the lambs. Four groups of pregnant ewes received concentrate containing 5.5, 9.9, 14.8, and 21.0 mg I/kg DM, respectively. Hay and molasses (containing 0.16 and 0.29 mg I/kg DM, respectively) were available ad libitum. The lambs were prevented from suckling for the first 24 h of life and were fed a fixed quantity of artificial colostrum in four feeds. At 24 h, the average plasma concentrations of immunoglobulin G in the lambs were 6.08, 5.06, 3.18 and 3.10 g/l for the 5.5, 9.9, 14.8 and 21.0 mg/kg groups, respectively. Supplementation with I was associated with higher levels of tri-iodothyronine and thyroxine in the lambs at birth. There was no effect of treatment on the plasma concentrations of insulin, cortisol, glucose or NEFA in the lambs. The concentration of dietary I that had an effect on the immunoglobulin concentration in the lambs is marginally above the levels added to commercial concentrate feeds; we tentatively identify 9.9 mg I/kg DM (approximately 9 mg I/ewe per day) as the upper safe limit of I supplementation according to the criterion of the concentration of immunoglobulin G in the plasma of lambs at 24 h after birth.

Effects of syringe material and temperature and duration of storage on the stability of equine arterial blood gas variables.

OBJECTIVES: To evaluate the consistency of partial pressures (P) of arterial oxygen (aO(2)), arterial carbon dioxide (aCO(2)) and pH measurements in equine carotid arterial blood samples taken into syringes made from three different materials and stored at room temperature or placed in iced water for measurement at three different times. STUDY DESIGN: Prospective observational study over 19 days. ANIMALS: Four clinically normal Thoroughbred or Thoroughbred-cross horses (three geldings, one mare, mean age 6.25 years, range 5-7 years). METHODS: Identical blood samples were taken on two separate occasions from the carotid arteries of the four horses into syringes made of glass, plastic and polypropylene. PaO(2), PaCO(2) and pH determinations were performed on blood from each syringe type at 10, 60 and 120 minutes post-sampling with samples stored at room temperature (approximately 20 degrees C) or in iced water (approximately 0 degrees C). Data were analysed by anova and a split plot model fitting syringe within horse X pair and time within temperature within syringe. RESULTS: Syringe material, storage temperature and time before analysis all had significant effects on PaO(2) (p < 0.001). PaCO(2) was unaffected by syringe material or storage temperature. However, over 120 minutes, storage duration significantly (p = 0.002) affected values. Temperature of storage and duration prior to analysis both significantly affected pH values (p = 0.005 and p < 0.001, respectively), but syringe material did not. Several significant interactions between these variables were noted. CONCLUSIONS: Equine arterial blood gas determination has a different sensitivity to storage conditions compared to other veterinary species. CLINICAL RELEVANCE: For accurate equine arterial blood analysis, PaO(2) samples need to be analysed within 10 minutes or taken into glass syringes, stored on ice and analysed at 2 hours post-sampling. PaCO(2) and pH measurements can be performed on samples stored in glass, plastic or polypropylene syringes at room temperature for up to 1 hour post-sampling.