Multiple Reaction Monitoring for the Accurate Quantification of Amino Acids: Using Hydroxyproline to Estimate Collagen Content.

Multiple reaction monitoring (MRM) mass spectrometry may be regarded as the gold standard methodology for quantitative mass spectrometry and has been adopted for the analysis of small molecules especially within the pharmaceutical industry. It can also be applied to the analysis of peptides and proteins and to measurement of the basic building blocks of proteins, amino acids. Here we describe the application of MRM mass spectrometry to the measurement of hydroxyproline after acid hydrolysis of various animal tissues. We show that measurement of hydroxyproline provides an accurate and reliable estimate of the collagen content of such tissues and may be a useful indicator of meat tenderness.

Predicting the effects of parasite co-infection across species boundaries.

It is normal for hosts to be co-infected by parasites. Interactions among co-infecting species can have profound consequences, including changing parasite transmission dynamics, altering disease severity and confounding attempts at parasite control. Despite the importance of co-infection, there is currently no way to predict how different parasite species may interact with one another, nor the consequences of those interactions. Here, we demonstrate a method that enables such prediction by identifying two nematode parasite groups based on taxonomy and characteristics of the parasitological niche. From an understanding of the interactions between the two defined groups in one host system (wild rabbits), we predict how two different nematode species, from the same defined groups, will interact in co-infections in a different host system (sheep), and then we test this experimentally. We show that, as predicted, in co-infections, the blood-feeding nematode Haemonchus contortus suppresses aspects of the sheep immune response, thereby facilitating the establishment and/or survival of the nematode Trichostrongylus colubriformis; and that the T. colubriformis-induced immune response negatively affects H. contortus This work is, to our knowledge, the first to use empirical data from one host system to successfully predict the specific outcome of a different co-infection in a second host species. The study therefore takes the first step in defining a practical framework for predicting interspecific parasite interactions in other animal systems.

Multiple reaction monitoring for the accurate quantification of amino acids: using hydroxyproline to estimate collagen content.

Multiple reaction monitoring (MRM) mass spectrometry may be regarded as the gold standard methodology for quantitative mass spectrometry and has been adopted for the analysis of small molecules especially within the pharmaceutical industry. It can also be applied to the analysis of peptides and proteins and to the measurement of the basic building blocks of proteins, amino acids. Here, we describe the application of MRM mass spectrometry to the measurement of hydroxyproline after acid hydrolysis of various animal tissues. We show that the measurement of hydroxyproline provides an accurate and reliable estimate of the collagen content of such tissues and may be a useful indicator of meat tenderness.

Overcoming macrocyclic lactone resistance in Haemonchus contortus with pulse dosing of levamisole.

The in vivo effect of dosing levamisole as a pulse release within an ivermectin (IVM) controlled-release device (CRD) was simulated by periodic dosing of levamisole to Haemonchus contortus-infected sheep already treated with an IVM CRD. The rationale for this treatment combination arises from the need to find alternative approaches to the treatment of gastrointestinal parasites in livestock in the face of increasing levels of anthelmintic resistance which is now widespread in Australia. Thirty merino sheep (4 months of age) were infected weekly with a mixture of susceptible and ivermectin resistant H. contortus beginning at Day 28. Twenty eight days after first infection, groups of 10 sheep were treated with IVM capsules alone, IVM capsules and an oral dose of levamisole (LEV) at Days 50 and 100 or no treatment. At pre-determined intervals, up to 126 days after treatment, faecal worm egg counts (FWEC) were determined and development rates of infective larvae (L3) cultured in faeces were measured. Haematological parameters and drug concentration in plasma were measured throughout the 100-day release period of the controlled-release device. Sheep were slaughtered at Day 135 for estimates of total worm burden. FWEC of sheep treated with IVM+LEV declined (99.9% reduction) after administration of oral LEV and were suppressed until Day 98. There was a significant difference (p<0.0001) in worm counts at slaughter between groups. The results demonstrate the potential advantage of combining a pulse of short-acting drug into the long-acting anthelmintic capsule to provide better parasite control than that achieved from the existing CRD treatment when IVM-resistant worms were present.

High-level ivermectin resistance in a field isolate of Haemonchus contortus associated with a low level of resistance in the larval stage: implications for resistance detection.

The IVPro isolate of Haemonchus contortus was isolated in 1999 after significant numbers of the parasite survived an ivermectin capsule treatment of grazing sheep acquiring a natural infection at Prospect, NSW, Australia. The isolate shows high-level resistance to ivermectin (faecal egg count is unaffected by ivermectin oral treatment at 0.2mg kg(-1)). The larval LC(50), as assessed by larval development assays (LDAs), is only approximately two-fold higher than several susceptible isolates, making it difficult to detect the resistance using larval LC(50) as an indicator. However, the isolate shows the presence of a small proportion of the population (<20%) able to develop at significantly higher drug concentrations than the susceptible isolates. Hence, if the IVPro and susceptible isolates are compared at the LC(99) level, the IVPro isolate is readily identifiable as resistant. This degree of distinction at the LC(99) allows the IVPro isolate to be identified as resistant by simply observing the highest drug concentration in the development assay at which some larvae develop relative to the susceptible isolates. Assessing the development assay using this criterion allows a distinction between IVPro and the susceptible isolates equivalent to 10-fold differences in drug concentration, greatly increasing the likelihood of detecting the resistant isolate in routine resistance tests. This study highlights the need to examine this aspect of LDAs in order to detect the type of resistance displayed by IVPro H. contortus.