Worms and welfare: Behavioural and physiological changes associated with gastrointestinal nematode parasitism in lambs.

Parasitism with gastrointestinal nematodes (GIN) is a worldwide issue impacting negatively on animal production, health, and welfare. Therefore, early diagnostic signs of parasitism are required to allow for timely interventions. The objective of this study was to evaluate the behavioural and physiological changes in lambs associated with GIN infection. We used 30, 8-month-old Romney-cross wethers, that were administered anthelmintics until faecal egg counts (FEC) were zero and housed in an indoor facility. The study lasted 9 weeks, which comprised a 3-week pre-treatment, and a 6-week treatment phase. Lambs were randomly assigned to one of two treatments (n = 15/treatment) trickle-dosed with: 1) 1500 infective third stage larvae (L3) three days/week for 6 weeks (27,000 total L3; challenged), or 2) water 3 days/week for 6 weeks (control). Within each pen there were 5 pairs of lambs (balanced for liveweight), with each pair comprising a challenged and control lamb. Blood, faecal, and saliva samples were collected 1 week pre-treatment and weekly for 6 weeks of treatment. Behaviour was observed (e.g., feeding, lying, standing) from video-camera recordings using scan sampling every 5 min for 8 h, 1 day pre-treatment and on the day immediately prior to physiological sampling across the 6-week treatment phase (7 days in total). Accelerometers were attached to each lamb to continuously monitor behaviour from 3 weeks pre-treatment and for the remainder of the study. Liveweight, body condition, faecal soiling and faecal consistency scoring were performed weekly as was lipidomic analysis of plasma samples. From week 2 of treatment, challenged lambs spent less time feeding and more time lying than control lambs until week 5 of treatment (P ≤ 0.01). At week 3 of treatment, elevated lipids (mainly triglycerides and phospholipids), loose faeces and faecal soiling around the anus were observed in challenged lambs compared with controls (P ≤ 0.05). From week 4 of treatment, FEC were elevated in the challenged compared to control lambs (P ≤ 0.05). There was also lower liveweight gain at 4 and 5 weeks of treatment in the challenged lambs compared with control lambs (P ≤ 0.05). These results show a clear timeline of changes in behaviour (e.g., feeding and lying), lipids such as triglycerides, and digestive function (e.g., faecal soiling) suggestive of GIN subclinical disease, which show promise for use in future studies on early identification of subclinical GIN parasitism in lambs.

Identification of Peptide Mimics of a Glycan Epitope on the Surface of Parasitic Nematode Larvae.

Phage display was used to identify peptide mimics of an immunologically protective nematode glycan (CarLA) by screening a constrained C7C peptide library for ligands that bound to an anti-CarLA mAb (PAB1). Characterisation of these peptide mimotopes revealed functional similarities with an epitope that is defined by PAB1. Mimotope vaccinations of mice with three selected individual phage clones facilitated the induction of antibody responses that recognised the purified, native CarLA molecule which was obtained from Trichostrongylus colubriformis. Furthermore, these mimotopes are specifically recognised by antibodies in the saliva of animals that were immune to natural polygeneric nematode challenge. This shows that antibodies to the PAB1 epitope form part of the mucosal polyclonal anti-CarLA antibody response of nematode immune host animals. This demonstrates that the selected peptide mimotopes are of biological relevance. These peptides are the first to mimic the PAB1 epitope of CarLA, a defined larval glycan epitope which is conserved between many nematode species.

Steady-state and non-steady state operation of counter-current chromatography devices.

Different variants of separation processes based on steady-state (continuous sample loading) and non-steady state (batch) operating modes of CCC columns have been analyzed and compared. The analysis is carried out on the basis of the modified equilibrium cell model, which takes into account both mechanisms of band broadening - interphase mass transfer and axial mixing. A full theoretical treatment of the intermittent counter-current chromatography with short sample loading time is performed. Analytical expressions are presented allowing the simulation of the intermittent counter-current chromatography separations for various experimental conditions. Chromatographic and extraction separations have been compared and advantages and disadvantages of the two methods have been evaluated. Further technical development of the CCC machines to implement counter-current extraction separations is considered.

Separation of patuletin-3-O-glucoside, astragalin, quercetin, kaempferol and isorhamnetin from Flaveria bidentis (L.) Kuntze by elution-pump-out high-performance counter-current chromatography.

Flaveria bidentis (L.) Kuntze is an annual alien weed of Flaveria Juss. (Asteraceae) in China. Bioactive compounds, mainly flavonol glycosides and flavones from F. bidentis (L.) Kuntze, have been studied in order to utilize this invasive weed, Analytical high-performance counter-current chromatography (HPCCC) was successfully used to separate patuletin-3-O-glucoside, a mixture of hyperoside (quercetin-3-O-galactoside) and 6-methoxykaempferol-3-O-galactoside, astragalin, quercetin, kaempferol and isorhamnetin using two runs with different solvent system. Ethyl acetate-methanol-water (10:1:10, v/v) was selected by analytical HPCCC as the optimum phase system for the separation of patuletin-3-O-glucoside, a mixture of hyperoside and 6-methoxykaempferol-3-O-galactoside, and astragalin. A Dichloromethane-methanol-water (5:3:2, v/v) was used for the separation of quercetin, kaempferol and isorhamnetin. The separation was then scaled up: the crude extract (ca 1.5 g) was separated by preparative HPCCC, yielding 12 mg of patuletin-3-O-glucoside at a purity of 98.3%, yielding 9 mg of a mixture of hyperoside and 6-methoxykaempferol-3-O-galactoside constituting over 98% of the fraction, and 16 mg of astragalin (kaempferol-3-O-glucoside) at a purity of over 99%. The pump-out peaks are isorhanetin (98% purity), kaemferol (93% purity) and quercitin (99% purity). The chemical structure of patuletin-3-O-glucoside and astragalin were confirmed by MS and ¹H, ¹³C NMR.

Probabilistic model for immiscible separations and extractions (ProMISE).

Chromatography models, liquid-liquid models and specifically Counter-Current Chromatography (CCC) models are usually either iterative, or provide a final solution for peak elution. This paper describes providing a better model by finding a more elemental solution. A completely new model has been developed based on simulating probabilistic units. This model has been labelled ProMISE (probabilistic model for immiscible phase separations and extractions), and has been realised in the form of a computer application, interactively visualising the behaviour of the units in the CCC process. It does not use compartments or cells like in the Craig based models, nor is it based on diffusion theory. With this new model, all the CCC flow modes can be accurately predicted. The main advantage over the previously developed model, is that it does not require a somewhat arbitrary number of steps or theoretical plates, and instead uses an efficiency factor. Furthermore, since this model is not based on compartments or cells like the Craig model, and is therefore not limited to a compartment or cell nature, it allows for an even greater flexibility.

Anthelmintic resistance in nematode parasites of cattle: a global issue?

Acceptable performance of grazing cattle frequently depends on the availability of effective broad-spectrum anthelmintics to remove, or prevent infection with, gastrointestinal nematodes. This control is increasingly threatened by populations of nematodes resistant to the most commonly used anthelmintics. Although this appears to have developed more slowly than in nematodes infecting small ruminants, the number of reports in the literature over the past five years suggests a rapidly escalating problem. This review discusses this literature, several issues unique to cattle parasitism and anthelmintics, and how previous research in small ruminants can improve the management of anthelmintic resistance in cattle.

The application of phage-displayed peptide libraries to ligand detection in eggs and larvae of Rhipicephalus (Boophilus) microplus.

The cattle tick, Rhipicephalus (Boophilus) microplus is an ectoparasite of cattle and is one of the major limiting factors in the use of Bos taurus cattle in tropical and subtropical countries. Current control relies heavily on chemotherapy with synthetic acaricides, which is threatened by the development of resistant tick populations. Novel approaches to target discovery in cattle ticks may provide alternative strategies for the control of these parasites. The value of phage-display technology in target discovery was assessed using late-stage (20 d) R. microplus eggs. Eight, 15-mer phage peptides were isolated which preferentially bound to the eggs, or to the larvae visible within. Western blot analyses indicated that the phage clones all bound to the same 16 kDa tick antigen. The results indicate the potential utility of phage-display in detecting 'unknown' cell surface targets on R. microplus, or indeed a range of other parasite species, which may be suitable targets for chemotherapy or vaccination strategies.

A new non-synchronous preparative counter-current centrifuge-the next generation of dynamic extraction/chromatography devices with independent mixing and settling control, which offer a step change in efficiency.

A new and significantly more robust design of non-synchronous coil planet centrifuge is introduced where the degree of mixing between two immiscible phases can be changed independently from the "g" field required to separate out the phases. A hypothesis that an optimum ratio between the speed of the bobbin and the speed of the rotor can be found to optimise the efficiency of the separation for a given force field is upheld for an intermediate polarity phase system. This paves the way for extensive further research to find the optimum non-synchronous conditions for a range of different phase systems that are desirable for the separation of large molecules, proteins and biologics but can tend to emulsify in the standard "J" type centrifuge systems currently available and routinely in use for aqueous organic phase systems. A step change of up to 30% in resolution and 90% in plate efficiency is demonstrated.

Multiple dual-mode countercurrent chromatography applied to chiral separations using a (S)-naproxen derivative as chiral selector.

Countercurrent chromatography (CCC) is a liquid-liquid chromatographic technique without a solid support. Several alternative elution modes can be applied to take advantage of the special nature of the liquid stationary phase. Among these dual-mode (DM) and multiple dual-mode (MDM) consist of switching alternatively between Reversed and Normal Phase operation during the experiment (once for DM and several times for MDM). In this paper, MDM has been applied to the chiral CCC separations of two racemic mixtures, (+/-)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5-dinitrobenzamide and N-(3,5-dinitrobenzoyl)-(+/-)-leucine, using (S)-naproxen N,N-diethylamide as chiral selector (CS). Although the behaviour of the two analytes differed, improved resolution factors were successfully obtained. Results are rationalized on the basis of the distinct partition behaviour of the CS/enantiomer complexes in the biphasic system.

Universal counter-current chromatography modelling based on counter-current distribution.

There is clearly a need for a model which is versatile enough to take into account the numerous operating modes and pump out procedures that can be used with counter-current chromatography (CCC). This paper will describe a universal model for counter-current chromatography based on counter-current distribution. The model is validated with real separations from the literature and against established CCC partition theory. This universal model is proven to give good results for isocratic flow modes, as well as for co-current CCC and dual flow CCC, and will likely also give good results for other modes such as intermittent CCC.

Advantages of a small-volume counter-current chromatography column.

Counter-current chromatography (CCC) works with a support-free liquid stationary phase. This allows for preparative separations and purifications. However, there are serious technical constraints because of the need to keep a liquid stationary phase in a column. Centrifugal fields are used. A new commercial hydrodynamic 18mL column made with a narrow-bore 0.8mm Teflon tubing was evaluated by comparing it with older hydrodynamic CCC columns and a similar 19mL column but made with 1.6mm Teflon tubing. A small-volume CCC column allows for reliable and fast solute partition coefficient determination. When resolution is required, both high efficiency and liquid stationary phase retention are needed. Unfortunately, these two requirements bear technical contradictions. A column coiled with a narrow tubing bore will provide a high chromatographic efficiency while a column containing wider tubing bore will achieve higher stationary phase retention. In all cases, increasing the magnitude of the centrifugal field also increases the stationary phase retention. The solution is to build centrifuges able to produce high fields that will provide acceptable liquid phase retention with narrow-bore tubes. The new 18mL 0.8mm tubing bore column is able to rotate as fast as 2100rpm generating a 240xg field. The two older CCC columns cannot compete with the new one. However, the small 19mL column with 1.6mm bore tubing can be useful when fast results are desired without top resolution.

Role of counter-current chromatography in the modernisation of Chinese herbal medicines.

This review focuses on the growing popularity of using counter-current chromatography (CCC), with its liquid stationary phase, as one of the prime methods for isolating compounds from Chinese herbal medicines (CHMs). 198 publications are reviewed covering 108 different plant species from 56 plant families. These describe the isolation of 354 different molecules across a wide range of polarities, chemical classes and molecular weights (in the range 100-1000 Da). The suitability of CCC for the separation of active compounds from CHM, the phase systems used, how CCC has developed in China, compounds isolated, CCC instrumentation, performance, operational issues and innovations, all supported by detailed cross-referencing, are described. It is concluded that CCC is making an increasingly important contribution to the modernisation of Chinese herbal medicines.

How to realize the linear scale-up process for rapid purification using high-performance counter-current chromatography.

This study used the isolation of six constituents from Selaginella tamariscina as an example to demonstrate how to achieve rapid and predictable linear scale-up processes in both normal- and reversed-phase high-performance counter-current chromatography. After systematic optimization of solvent systems, sample concentration, sample loading volume, rotation speed and flow rate on the analytical Mini-DE centrifuge, the optimized parameters obtained were directly transferred to the preparative Midi-DE centrifuge, with nearly the same purities, resolutions and elution times but with 50 times the throughput. Amentoflavone (446.7 mg, 97.8%), robustaflavone (21.6 mg, 89.4%), bilobetin (80.7 mg, 92.7%), hinokiflavone (15.1 mg, 85.5%), isocryptomerin (34.8 mg, 89.6%) and an apigenin-diglucoside (46.3mg, 96.4%) were obtained with amounts and purities shown in parentheses as analysed by HPLC. The process, therefore, offers an efficient and rapid method of obtaining sufficient quantities of target compounds with significantly increased throughput after a linear scale-up.

Review of centrifugal liquid-liquid chromatography using aqueous two-phase solvent systems: its scale-up and prospects for the future production of high-value biologicals.

Future challenges in the field of bioprocessing include developing new downstream processes for the purification and manufacture of protein-based medicines to relieve the predicted bottleneck that may occur as a result of increasingly high titers obtained from fermentation processes. This review considers recent developments in centrifugal liquid-liquid partition chromatography using aqueous two-phase solvent systems, a gentle host medium for biologicals, and the prospect for scale-up and eventual manufacture of high-value pharmaceutical products.

Intestinal, hepatic, splanchnic and hindquarter amino acid and metabolite partitioning during an established Trichostrongylus colubriformis infection in the small intestine of lambs fed fresh Sulla (Hedysarum coronarium).

Increased partitioning of amino acids (AA) from skeletal muscle to the intestine and immune system during parasitic infection may be the cause of poor growth in parasitised animals. The effect of an established Trichostrongylus colubriformis infection (6000 L3 T. colubriformis larvae for 6 d (n 5) or kept as parasite-free controls (n 6)) on AA fluxes across the mesenteric-drained viscera, portal-drained viscera (PDV), liver, total splanchnic tissues (TSP) and hindquarters were determined in lambs fed fresh Sulla (Hedysarum coronarium; 800 g DM/d) 48 d post-infection. The lambs were infused with rho-aminohippuric acid (PAH; 723 mg/h) into the mesenteric vein for 8 h to measure TSP plasma flow. Concurrently, indocyanine green (ICG; 14.6 mg/h) was infused into the abdominal aorta to measure plasma flow across the hindquarters. Blood was continuously collected from the mesenteric, portal and hepatic veins, vena cava and the mesenteric artery and plasma harvested. PAH, ICG, AA, metabolite and insulin concentrations were measured. Intestinal worm burdens on day 48 post-infection were higher in the infected lambs (P 0.10). There was a 28 % reduction in the release of AA from the PDV of infected lambs (P < 0.05). The uptakes of most AA were similar in the liver; however, there was increased uptake (P < 0.10) of AA by the TSP of infected lambs. Despite this reduction in AA availability at the liver, there was no effect of parasitic infection on AA uptake across the hindquarters (P < 0.05).

Recent progress on the industrial scale-up of counter-current chromatography.

The pharmaceutical industries are looking for rapid methods of purification and predictable scale-up for their drug development process that will cut their costs and enable them to reduce the time to market. In this paper, recent progress is reviewed in the development and demonstration of two types of industrial scale centrifugal liquid-liquid chromatography: hydrostatic and hydrodynamic. Industrial scale hydrostatic processes by Partus Technologies and Armen Instrument are just emerging. Results demonstrating scalability are presented for hydrodynamic processes by Dynamic Extractions. The review concludes that the time is now right, with this appropriate commercial support, for high performance counter-current chromatography to emerge as a major enabling technology for industry.

Counter-current chromatography separation scaled up from an analytical column to a production column.

An analytical separation was performed on an analytical J-type counter-current chromatography (CCC) instrument using a 5.4 ml column, with a 1 ml/min mobile phase flow rate. This separation had a resolution of 0.69 and was achieved in 10 min. The same separation was performed using two 2300 ml columns connected in series at a flow rate of 850 ml/min using a production scale J-type centrifuge. This production scale separation was also obtained in 10 min with a resolution of 0.71. This represents an 850 times increase in productivity. This paper presents these separations and the underlying scale up theory.

Rapid purification and scale-up of honokiol and magnolol using high-capacity high-speed counter-current chromatography.

In this paper, a rapid separation approach has been developed using high-capacity high-speed counter-current chromatography (high-capacity HSCCC) to isolate and purify honokiol and magnolol, which are the main bioactive constituents from Houpu. The optimization of the solvent selection process, sample loading volume and flow rate is systematically studied using analytical high-capacity HSCCC. The optimized parameters obtained rapidly at analytical scale were used for a 1000 x scale-up preparative run using pilot scale high-capacity HSCCC in a MAXI-DE centrifuge. A crude sample of 43 g was successfully separated and the fractions were analysed by high-performance liquid chromatography (HPLC). This large scale preparative single step run yielded 16.9 and 19.4 g of honokiol and magnolol with purities of 98.6 and 99.9%, in only 20 min. This is the first time that high-performance counter-current chromatography has been used to purify multiple gram grade bioactive compounds in less than 1h and at such high concentrations of final products (10.8 g/l for magnolol and 7.0 g/l for honokiol).

Whole-body valine and cysteine kinetics and tissue fractional protein synthesis rates in lambs fed Sulla (Hedysarum coronarium) and infected or not infected with adult Trichostrongylus colubriformis.

Poor growth during parasitic infection may be due to a redistribution of amino acids away from skeletal muscle protein synthesis to the intestinal site of infection. The effect of a Trichostrongylus colubriformis infection on whole-body amino acid kinetics and tissue fractional protein synthesis rates were determined in lambs fed fresh Sulla (Hedysarum coronarium; 800 g DM/d). Lambs were dosed with 6000 L3 Trichostrongylus colubriformis larvae daily for 6 d (n 6) or kept as parasite-free controls (n 6). On day 45 post-infection, the lambs received an intravenous injection of 2H2O and infusions (8 h) of [35S]sulphate to measure the size of the whole-body water and sulphate pools, respectively. On day 48, the lambs were continuously infused for 8 h with [3,4-3H]valine into the jugular vein as well as with [1-13C]valine and [35S]cysteine into the abomasum. After the 8 h infusions, the lambs were killed and tissue samples collected from the duodenum, ileum, mesenteric lymph nodes, liver, spleen, thymus, muscle and skin. Feed intake (769 v. 689 (sd 47) g DM/d) was not affected by infection, whereas liveweight gains (50 v. -50 (sd 70) g/d) were lower and intestinal worm burdens (240 v. 18,000 (sd 7000) worms) higher in the infected lambs. Parasitic infection increased the fractional protein synthesis rates in the small intestine, mesenteric lymph nodes and liver but did not affect skin and skeletal muscle fractional protein synthesis rates during the established parasitic infection.