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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors wish to declare no competing interests. This study was funded by the New Zealand Ministry for Business, Innovation and Employment. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The mechanisms underlying resistance to challenge by gastrointestinal nematode parasites in sheep are complex. Using DIGE, we profiled ovine lymph proteins in lambs with host resistance (R), resilience (Ri) or susceptibility (S) to a daily trickle challenge with the nematode Trichostrongylus colubriformis. Efferent intestinal lymph was collected prior to infection (day 1) and on days 5 and 10 post-infection. Eight proteins identified by LC-MS/MS, showed differences relating to host genotype. Of these, Serpin A3-3 and Serpin A3-7 have not been reported previously in the lymph proteome. Three acute phase proteins showed significant differences relating to interactions between breeding line and parasite challenge, including complement C3β, C3α and haptoglobin (Hp) β. In the R lambs C3α was significantly up regulated (P<0.05) on day 10, while in the Ri lambs Hp β was significantly down regulated (P<0.05). In the S lambs, levels of C3β were up regulated and levels of Hp β down regulated (both P<0.05) on day 10. Hence we demonstrate that acute phase inflammation proteins contribute to differences in the innate immune response of sheep to challenge by T. colubriformis. The findings may lead to the development of new approaches to combat nematode infestations in sheep production systems., Biological Significance: Breeding lines of sheep with resistance (R), resilience (Ri) or susceptibility (S) to nematode infections provide an experimental model to examine the biological mechanisms underlying the ability of some sheep to expel worms and remain healthy without the use of an anthelmintic. Using proteomics we identified differences in the expression of acute phase lymph proteins in the R, Ri and S lambs. The results will assist the development of alternative control strategies to manage nematode infections in livestock., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The effect of condensed tannins (CT) extracted from forage plants from Botswana on the free-living stages of a number of species of gastrointestinal nematode parasites derived from infected sheep were investigated using in vitro assays. Fresh samples of five different plants (Viscum rotundifolium, Viscum verrucosum, Tapinanthus oleifolius, Grewia flava and Ipomoea sinensis) were collected over two summers (February 2009 and 2010). Fractionation of each crude extract on a Sephadex LH-20 column yielded low molecular weight phenolics and CT-containing fractions. The effect of each purified CT fraction on parasites was evaluated using either egg hatch, larval development or larval migration inhibition assays. Three gastrointestinal nematode species (Haemonchus contortus, Trichostrongylus colubriformis and Teladorsagia circumcincta) derived from infected sheep were evaluated in the study. CT from V. rotundifolium and I. sinensis fractions from samples collected in 2009 and 2010 did not inhibit larval development. However, CT isolated from V. verrucosum, T. oleifolius and G. flava collected in 2009 completely inhibited the development of all parasite species. These CT fractions were more potent in inhibiting larval development of H. contortus than fractions from the same plant species collected in 2010. However, a slight effect on larval migration was observed with some CT extracts. The results suggest that CT extracts of some forage plants from Botswana have anti-parasitic properties in vitro, and that further research is required to determine any in vivo efficacy from feeding the plants to goats in a field situation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The glycolipid CarLA (carbohydrate larval antigen) is present on the epicuticle of the infective-stage larvae of gastrointestinal nematode parasites infecting livestock. The molecule is lost from the surface of the larvae in the few days post-ingestion by a host animal, and the resulting anti-CarLA antibody response has been demonstrated to be protective in vivo. Both the anti-CarLA response, and anti-parasite immunity in general, are slow to develop, and several months of natural exposure to ingested larvae is required. The current study was designed to provide information on how the anti-CarLA response develops, and focuses on the initial recognition of the molecule by human monocyte derived dendritic cells (mdDC) in vitro. Immunofluorescence and flow cytometry demonstrated that mdDC recognise and internalise both the purified and the native form of CarLA, in the case of the latter once it is shed from the larval surface. However, the recognition of CarLA did not result in classical maturation of DC, while there was only transient or minor up-regulation of CD86, CD83, HLA-DR and CD40. Exposure of mdDC to purified CarLA resulted in the increased production of the pro-inflammatory cytokines IL-6 and to a lesser extent of IL-8 and TNF-α, and a reduced production of the anti-inflammatory cytokine IL-1RA. CarLA therefore has little ability to mature and functionally alter monocyte derived dendritic cell function., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Infection with gastrointestinal nematodes (GIN) is a major constraint on the productivity of grazing livestock. The development of selection methods to quickly and accurately identify animals capable of developing an effective natural immunity to infection would contribute to the development of sustainable worm control programs. A carbohydrate larval surface antigen (CarLA), present on the infective-stage larvae (L3) of all trichostrongylid nematodes, is a target antigen for host antibody (Ab). The levels of various Ab isotypes in serum and/or saliva of field-grazed lambs were assessed by ELISA, and Ab titres compared with parasite faecal egg counts (FECs) and a range of animal productivity parameters. Levels of anti-CarLA IgA in saliva proved to be the most heritable (h(2)=0.3), and had the closest genetic correlation with FEC (r=-0.5). Those animals identified as having 'high levels' of anti-CarLA IgA typically have 20-30% lower FEC than animals with low or undetectable titres. Furthermore, animals with 'high levels' of anti-CarLA IgA tend to have improved growth rates post-weaning, and have no tendency for increased breech-soiling. The assay performed well regardless of parasite genera present on pasture. The saliva assay has a number of key practical advantages over the use of FEC for selection purposes: animals can be identified without a requirement to withhold anthelmintic treatment; sampling is rapid and easy and there is a significantly reduced barrier to adoption within the farming community. Measurement of anti-CarLA IgA in saliva by ELISA offers a practical, rapid and easy method of selecting for natural immunity to GIN in sheep., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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., (Copyright 2010 Elsevier B.V. All rights reserved.)

To determine the impact of anthelmintic resistance on the productivity of sheep grazed on pasture in a temperate climatic zone, 14 groups each of 20 lambs were grazed on pasture on which benzimidazole-resistant parasites had been detected previously, then treated every 28 days - seven groups with a benzimidazole anthelmintic (albendazole) and seven with monepantel, a member of a new anthelmintic action family which was assumed in advance to be completely effective in removing all established worms. Faecal egg counts and larval differentiation demonstrated the presence of albendazole resistance, predominantly in Teladorsagia circumcincta but also in Trichostrongylus spp. By days 84 and 112, egg counts were significantly higher in the albendazole-treated animals than in those treated with monepantel. The presence of anthelmintic resistance resulted in a reduction in live-weight of 2.8 kg, a significant increase in breech-soiling and a significant reduction in body condition score. Fourteen animals from each treatment were necropsied at a commercial abattoir and carcase weights and standard quality parameters recorded; there was a reduction in carcase weight of 2.8 kg in the albendazole-treated animals, and a difference in the carcase grades within each group. These measurements were used to calculate that the presence of anthelmintic resistance resulted in a 14% reduction in carcase value., (Copyright 2010 Elsevier B.V. All rights reserved.)

Separation of large bioactive molecules such as proteins, DNAs and RNAs using aqueous two-phase systems (ATPSs) and liquid-liquid partition-based counter-current chromatography (CCC) can avoid risks of sample loss and denaturation, and greatly reduce processing time. We have constructed toroidal columns (length 26-140 m, column volume 51-280 ml, bore size 1.6 mm) suitable for mounting onto a commercially available preparative CCC apparatus. With the use of an ATPS containing 12.5% (w/w) PEG1000 and 12.5% (w/w) K(2)HPO(4) and at a rotational speed of 800 rpm for the rotor of the CCC device, the lower phase (i.e. the phosphate-enriched phase) has been used as the mobile phase and a pair of proteins, myoglobin and lysozyme, as model proteins for demonstrating the separation capability of the CCC system. For a toroidal column with a length of 53.5 m and a column volume of 107.5 ml, and operated for the Coriolis force parallel flow mode at 0.62 ml/min, protein sample loading (containing 2.2 mg/ml myoglobin and lysozyme, respectively) at 1.7% and 7.4% to the column volume led to peak resolution (with theoretical plate number TP and stationary phase retention S(f) shown in the parenthesis) of R(s)=1.5 (N=211 and N=113 TP for myoglobin and lysozyme, respectively, and S(f)=45.0%), and R(s)=1.4 (218 and 152 TP, and S(f)=34.0%). However, further increase of the loading to 13% failed to separate the two proteins. Although proteins eluted at positions predictable from the distribution coefficients, they showed broader peaks when compared with small dipeptides under identical CCC operating conditions. This confirms that the molecular weight of the partitioned species is an important factor causing peak broadening on CCC chromatograms. These results paved the way for further scaling-up toroidal CCC columns for processing larger quantities of samples containing large biomolecules., (2010 Elsevier B.V. All rights reserved.)

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., (Copyright 2009 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

The effects of an established Trichostrongylus colubriformis infection on amino acid (AA) absorption from the small intestine and their availability to other tissues were determined in lambs 48 days post infection. The lambs were fed fresh Lucerne (Medicago sativa; 800 g dry matter (DM)/day) and dosed with 6000 L3 T. colubriformis larvae for 6 days (n = 5) or kept as parasite free controls (n = 6). Faecal egg production was monitored every second day from day 22 to day 48. A nitrogen (N) balance was conducted on days 35 to 43 after infection, and digesta flow and AA concentration measurements were made on day 44. On day 48 after infection, blood was continuously collected from the mesenteric artery and vein, plasma harvested and AA concentrations measured. Faecal egg production peaked on the 26th day after infection (P < 0.001) and intestinal worm burdens on day 48 were greater (P < 0.001) in the infected lambs. Feed intake and liveweight gain were similar (P > 0.10) between control and infected lambs. Digestibility and flow of DM and N through the digestive tract were also unaffected (P > 0.10) by parasite infection. Despite a trend towards higher abomasal AA flux in the parasitised lambs (P < 0.10), apparent AA absorption from the small intestine and AA availability to other tissues were unaffected (P > 0.10) by infection. These results suggest that an established parasite infection had little effect on the intestinal absorption and availability of AA to other tissues in lambs fed fresh Lucerne.

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.

The scaling up of the separation of two proteins with an aqueous two-phase system (ATPS) from 176 mg with a 500 ml laboratory scale centrifugal partition chromatography (CPC) column to 2.2g with a 6.25 litre pilot-scale column is presented. A model sample system of a mixture of lysozyme and myoglobin was chosen for this study using an ATPS system comprising 12.5% (w/w) PEG-1000:12.5% (w/w) K2HPO4. It was found that the maximum sample concentration possible without precipitation was 2.2mg/ml for each constituent. The optimisation of rotor speed, mobile phase flow rate and sample loading was performed on a laboratory-scale device. It was found that a centrifuge speed of 2000 rpm (224 'g'), 10 ml/min mobile phase flow rate with a 43 ml (10% of active column volume) sample volume gave optimum operating conditions. This was linearly scaled up to pilot scale by increasing mobile phase flow rate, fraction size and sample loading in the ratio of the system capacities (i.e. 12.5:1). Flow rate was therefore increased from 10 ml/min to 125 ml/min, fraction size from 10 ml to 125 ml and sample loading from 43 ml to 500 ml. Rotor speed however was reduced from 2000 rpm on the laboratory device to 1293 rpm on the pilot-scale device to maintain the same 224 'g' field in each chamber, as the pilot-scale CPC unit has a larger rotor radius than the laboratory one. Resolution increased from Rs=1.28 on the 500 ml rotor to Rs=1.88 on the 6.25 litre rotor, giving potential throughputs in batch mode of over 40 g/day.

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).

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.

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.

Stationary phase retention in a synchronous coil planet centrifuge or high-speed counter-current chromatography (CCC) relies on the interplay of hydrostatic (tangential and normal centrifugal) and hydrodynamic (Archimedean screw and mobile phase drag) forces. By offering a set of quantitative or semi-quantitative theoretical frameworks, this work has resolved fundamental questions such as "in the absence of mobile phase flow, how is the distribution of the two phases in a CCC column determined?" and "for Type-J CCC, do the helical and the spiral columns lead to similar performance?"

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.

Retention properties of polyethylene glycol-phosphate aqueous two-phase systems in a spiral coil (5 mm I.D.) on Type-J synchronous counter-current chromatographic devices have been compared for the elution mode where the lower phase is the mobile phase and flows from the inside head terminal. This was achieved with the aid of digital imaging under stroboscopic illumination, an image analysis and measurement of the displaced volume of the stationary phase. For the spiral coil, high and stable stationary phase retention at mobile phase flow rates up to 64 ml/min has been obtained. Wave-like disturbance of the interface near the proximal point was observed and analyses have been made for possible use in protein separation.

In dual-flow counter-current chromatography (DF-CCC), the two immiscible liquids are flowing in opposite directions in the coil. The method allows for the continuous separation of two solutes. In this study a numerical model was developed to allow for the detailed investigation of flow in such columns. The mesh model of the presented DF spiral column was developed in line with an existing experimental model. The paper presents results during the early filling stages for different rotation directions. These clearly illustrate the performance of the developed model by (1) confirming the importance of flowing the lighter phase from tail to head and the heavier phase from head to tail and (2) by visualising mixing waves and the recognised back and forth "swish-swash" motion as present in CCC in that operating mode.

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).

Accurate quantification with real-time PCR requires the use of stable endogenous controls. Recently, there has been much debate concerning the stability of commonly used reference or housekeeping genes. To address this concern, a number of statistical approaches have been designed to analyse data and assist in determining the most appropriate reference genes for experimental comparisons. In this study, three programs, BestKeeper, Norm Finder, and geNorm were used to assess four candidate reference genes: 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acidic ribosomal protein large (RPLP0) and beta-actin, for use in expression profiling of individuals from divergent cattle genotypes subject to parasitic challenge with the cattle tick Boophilus microplus. Results demonstrated beta-actin and GAPDH were the most suitable reference genes in blood and could be used either individually or combined as an index to normalise data. RPLP0 was identified as the least stable gene, while 18S rRNA was omitted as being too highly expressed. As the recommendations on the most suitable reference genes varied between the programs, it is recommended that more than one should be utilised, to ensure the most robust experimental tools are selected.

A new generation of high performance coil planet centrifuges is now making it possible to realize the enormous potential of liquid-liquid partition chromatography for the rapid and predictable scale up of separation processes. This paper uses a separation of flavonoids to demonstrate how rapid fractionations can be obtained at high flow rates with limited loss of resolution and with significant increase in throughput. Furthermore, it is shown that chromatograms at various flows can be modeled so that optimum conditions can be rapidly assessed.

Analytical Milli high-speed counter-current chromatography (HSCCC) was used for the selection and optimization of the two-phase solvent system to separate flavonoids from the extracts of the seeds of Oroxylum indicum. The optimum solvent system obtained from Milli-CCC was also the best solvent system for preparative HSCCC and led to the successful separation of two crude flavonoids from the seeds of O. indicum by Lab/Prep (laboratory preparative) HSCCC using different sized coils. Four flavonoids were isolated by preparative HSCCC: baicalein-7-O-diglucoside (25.0 mg, 92% purity), baicalein-7-o-glucoside (50.4 mg; 95% purity), baicalein (75 mg; purity 98%) and chrysin (100 mg; purity 98%).

In countercurrent chromatography (CCC) both stationary and mobile liquids undergo intense mixing in the variable force field of a coil planet centrifuge and the separation process, like the separation in conventional solvent extraction column, is influenced by longitudinal mixing in the phases and mass transfer between them. This paper describes how the residence time distribution (or the elution profile) of a solute in CCC devices and the interpretation of experimental peaks, can be described by a recently developed cell model of longitudinal mixing. The model considers a CCC column as a cascade of perfectly mixed equal-size cells, the number of which is determined by the rates of longitudinal mixing in the stationary and mobile phases. Experiments were carried out to demonstrate the validation of the model and the possibility of predicting the partitioning behaviour of the solutes. The methods for estimating model parameters are discussed. Longitudinal mixing rates in stationary and mobile phases have been experimentally determined and experimental elution profiles are compared with simulated peaks. It is shown that using the cell model the peak shape for a solute with a given distribution constant can be predicted from experimental data on other solutes.

Sixty-four Romney ewe lambs were allocated to 12 groups on the basis of liveweight. Four groups (n=5) were administered oral ivermectin (IVM), 4 (n=6) oral moxidectin (MOX) and the remaining 4 (n=5) controlled-release capsules containing IVM (IVM-CRCs). Nine and 10 days later, the groups within each treatment type were challenged with infective-stage larvae (L3) of 1 of 4 different isolates of Teladorsagia circumcincta (two doses each of 5000 L3). The first of these (S) was known to be anthelmintic-susceptible; the second (OR) was a multiple anthelmintic-resistant strain recovered from the field following therapeutic failure of both ivermectin and moxidectin and subsequently maintained in the laboratory without further anthelmintic selection; the third (R) was derived from OR but had been passaged for five generations indoors with each generation being screened with all three broad-spectrum anthelmintic classes; and the fourth (RxS) was an F1 cross between the R and S isolates. As anticipated, because of its limited residual activity, IVM had no significant effect on the establishment, 9 and 10 days post-treatment, of any of the parasite isolates. In contrast MOX, which has greater residual activity, was highly effective at preventing the establishment of the S isolate but showed no significant effect against the OR, R or RxS isolates. The IVM-CRC was also highly effective at preventing the establishment of the S isolate and furthermore it significantly reduced establishment of both the OR and RxS isolates, although it had no significant effect against the R isolate. The results suggest that with respect to the establishment of T. circumcincta L3s following anthelmintic treatment, macrocyclic lactone (ML) resistance is effectively a dominant trait in the presence of MOX, while it behaves as a partially dominant/recessive trait under treatment with IVM-CRCs. The potential implications of this finding in relation to selection for ML resistance in T. circumcincta are discussed.

Biosynthesis of polyketide antibiotics, such as erythromycin A (EA), can result in the formation of analogues of the main compound that are chemically and structurally extremely similar. The large-scale purification of these antibiotics by conventional high-performance liquid chromatography (HPLC) can be prohibitively expensive due to the large volume of both solvent and adsorbent required. This study examines the feasibility of using a novel pilot-scale countercurrent chromatography (CCC) machine as an alternative to HPLC. CCC is a low-pressure (typically <4000 kN m(-2)) liquid-liquid chromatographic technique that allows the separation of solutes on the basis of their partitioning between two immiscible liquid phases. The effects of mobile phase flow rate, column rotational speed, and sample injection volume on the attainable yield and purity of EA were investigated. Our results show that, at a mobile phase flow rate of 40 mL min(-1), a rotational speed of 1200 rpm, and an injection volume of 100 mL (10 g total erythromycin), EA could be satisfactorily fractionated with a purity of approximately 92% (w/w) and a recovery yield of approximately 100% (w/w). The total solute throughput was estimated to be 0.41 kg day(-1). More importantly, we demonstrated simple and predictive linear scale-up of the CCC separation based on data obtained from a single laboratory-scale CCC chromatogram, and verified this experimentally. The retention time and peak width of the target compound at the pilot scale could be predicted to within 4% for operation at a range of mobile-phase flow rates and injection volumes. This predictable nature of CCC separations, unlike HPLC methods, can greatly reduce process development times and enable a complete process-scale operating scenario to be planned., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 640-649, 2003.)

Challenge with an equal mix of drug-resistant and drug-susceptible larvae of Teladorsagia circumcincta resulted in infections in groups of lambs (n = 6) either untreated or given controlled-release capsules, containing either albendazole or ivermectin. Lambs treated with albendazole capsules contained similar numbers of adult worms at necropsy to the other groups but had no detectable faecal egg count. Animals treated with ivermectin capsules had similar worm burdens and faecal egg counts to the control group but the worms had significantly higher numbers of eggs in utero. These results provide evidence for suppression of egg production by both anthelmintic treatments. The observation that albendazole caused a significant reduction in the developmental success of parasite eggs also has implications for the use of faecal egg count as an indicator for pasture contamination with resistant parasites. In two further groups of lambs, either untreated or given albendazole capsules, treatment caused a significant reduction in egg count and adult worm burden of Trichostrongylus colubriformis. No significant effects were observed on in utero egg counts or egg viability and the apparent effect on the number of eggs produced in faeces per adult female was not significant (p = 0.077). There was, therefore, no evidence that albendazole controlled-release capsules caused suppression of egg output in this species.