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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.)

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.)

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.)

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.

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).

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.

Eighty-eight lambs were allocated to one of four groups which were then dosed with 10,000 infective-stage larvae (L3) of one of four populations of Ostertagia circumcincta; the first (S) was an isolate known to be anthelmintic-susceptible; the second (OR) was a multiple anthelmintic-resistant isolate which had been 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 in the laboratory with each generation being screened with all three broad-spectrum drench families; the fourth (R x S) was an F1 cross between the S and R isolates. On patency, each of the four infection groups was sub-divided into five treatment groups, one of which received no anthelmintic while the others were administered either oral ivermectin (IVM-oral), controlled-release capsules containing ivermectin (IVM-CRCs), oral moxidectin (MOX-oral) or injectable MOX (MOX-inj). Neither formulation of IVM reduced FEC in the R, R x S and OR infected lambs compared to their untreated controls, but significant reductions were observed in all cases following MOX-oral or MOX-inj treatment. Similarly, neither IVM formulation significantly reduced the numbers of R or R x S worms compared to their untreated controls, although the numbers of OR worms were reduced in both cases (P<0.05). Direct comparisons of efficacy across the isolates, however, indicated that neither formulation was any more effective against R x S or OR worms than against the more highly selected R worms. In contrast, both MOX formulations significantly reduced worm numbers of all the resistant isolates compared to their respective untreated controls; furthermore, worm burdens of R x S were reduced significantly more than burdens of R (P<0.05). Reductions in OR burdens, which were intermediate between the two, did not differ significantly from either. The results are consistent with published work on Haemonchus contortus, which suggests that macrocyclic lactone (ML) resistance is expressed as a dominant trait under treatment with IVM. However, these data differ from the H. contortus studies in suggesting that ML resistance in O. circumcincta may effectively be rendered incompletely dominant or recessive by treatment with MOX.

A recent paper by Du et al. [J. Chromatogr. A, 835 (1999) 231] showed a very good correlation between the retention of stationary phase and the square root of mobile phase flow (F(1/2)) for 12 different phase systems in counter-current chromatography. This paper shows there is a relationship between the above retention and the linear velocity of the mobile phase. In this way, Du et al.'s results can be related to the kinematics of the mobile phase flow in the tubing. This will open the door for further engineering analysis of this fluid dynamic phenomenon.

One of three groups of sheep was challenged twice-weekly with infective-stage larvae (L3) of the sheep parasite O. circumcincta, another with the cattle parasite O. ostertagi while the third received no larval challenge. Positive faecal egg counts (FEC) and a rise in plasma pepsinogen levels were observed only in those animals given O. circumcincta. Anti-O. circumcincta L3 IgG titres were rapidly elevated during parasite challenge with either O. circumcincta or O. ostertagi. Throughout the experiment, no rise in anti-adult IgG titres or eosinophil numbers was observed in peripheral blood in any group. On evidence of self-cure of the trickle-infection, determined by a reduction in FEC, all groups were drenched and challenged with 15,000 O. circumcincta L3. No effect of previous challenge on parasite establishment or FEC was observed, although egg viability was significantly reduced in both groups given prior challenge. Significant differences in adult female worm length were observed between groups. Those recovered from animals previously challenged with O. circumcincta were shorter than from those given O. ostertagi which were in turn shorter than those from previously unchallenged animals. In utero egg counts were significantly lower in worms from animals previously challenged with O. circumcincta than in those from unchallenged control animals. The results indicate that a level of immunity to O. circumcincta can be conferred by exposure to O. ostertagi.

The effect of albendazole (ABZ)-capsule (CRC) administration on parasite establishment and immunity to ABZ-resistant (RES) and -susceptible (SUS) T. colubriformis was measured in Romney lambs. During 12 weeks of twice-weekly dosing with 3000 parasite larvae (L3), eggs were observed in faeces from CRC-treated and untreated lambs given RES L3, but not CRC-treated lambs given SUS L3. Following the period of trickle challenge all lambs were drenched and, 1 week later, dosed with 20000 SUS L3. Resulting worm burdens were higher in control lambs than in those previously treated with CRCs and challenged with SUS, which in turn were higher than those in the CRC-treated or -untreated lambs previously challenged with RES L3. During the period of trickle challenge, the number of peripheral eosinophils and titres of anti-L3 and anti-adult antibody were raised only in those groups given RES L3. There was no effect of CRC administration. Following drench and challenge, antibody titres and eosinophil numbers increased in the control animals but not in those groups which had received previous trickle infection. The results demonstrate that the larval challenge alone resulted in incomplete though substantial protection against subsequent parasite challenge. The use of CRCs may potentially impact on subsequent animal performance and selection for anthelmintic resistance through a reduced level of immunity.

The transport of isometamidium chloride (Samorin) in Trypanosoma congolense which were either sensitive or resistant to this widely used trypanocide was studied in vitro. Significantly lower amounts of drug were accumulated over time by resistant than by sensitive trypanosomes. While no direct evidence could be obtained, indirect observations implied the involvement of an increased efflux of drug from the resistant trypanosomes. In both the resistant and sensitive parasites, drug transport was found to be mediated by an energy-dependent, specific process, presumably receptor-mediated. However, the specificity of the putative receptors was altered in the drug-resistant parasites. It is proposed that an alteration or replacement of a specific receptor in isometamidium chloride-resistant T. congolense results in an increased efflux of the drug and that this increased efflux at least partially mediates the reduction in sensitivity to the compound.

Isometamidium chloride (Samorin, RMB, England) is a widely used and highly effective trypanocide for the treatment of bovine trypanosomiases. However, the appearance of isometamidium-resistant populations of T. congolense in Africa makes it necessary to develop methods for the rapid and reliable detection of drug resistance in the laboratory. Currently available tests are time-consuming and/or expensive. In the present study, the short-term in vitro incubation of trypanosomes in a range of isometamidium concentrations and the infectivity of the parasites in mice has been assessed. A series of T. congolense isolates were used which were known to differ in their in vivo sensitivity to the drug. The results showed a close correlation between the known level of resistance and the capability of trypanosomes to remain infective after incubation in isometamidium. Thus isolates displaying a high level of resistance in vivo remained infective following incubation in higher concentrations of drug. This assay may provide a simple and reliable method for detecting drug resistance in T. congolense.

An investigation was conducted on the therapeutic and prophylactic activity of isometamidium chloride (SamorinR) in Boran (Bos indicus) cattle against a Trypanosoma congolense clone, IL 3270. This clone was derived, without drug selection, from a stock originally isolated in Burkina Faso and has previously been shown to be resistant to isometamidium in both cattle and mice using an infection and treatment regimen. A group of 5 cattle were treated intramuscularly with 1.0 mg kg-1 isometamidium chloride and 28 days later challenged with Glossina morsitans centralis infected with T. congolense IL 3270. All 5 cattle and 17 untreated cattle challenged on the same day became parasitaemic by day 16 post challenge, indicating that prophylaxis did not extend to 28 days post treatment. The cattle were then treated with isometamidium chloride at one of the following doses and by different routes of administration; 1.0 or 2.0 mg kg-1 intramuscularly, 0.25, 0.5, 0.75 or 1.0 mg kg-1 intravenously. Infections relapsed in all cattle at an interval of 12-21 days following treatment, with the exception of those treated with 2.0 mg kg-1 intramuscularly in which the development of relapse infections was delayed. Similar studies were also conducted with a highly sensitive clone of T. congolense, IL 1180. Infections in cattle with this clone were eliminated by intravenous treatment with 0.25 mg kg-1 isometamidium chloride or intramuscular treatment with 0.5 mg kg-1 isometamidium chloride. Thus, although intravenous administration of isometamidium eliminated a fully sensitive infection, treatment by this route appeared not to enhance the therapeutic efficacy of the drug in the treatment of a T. congolense clone which expresses a high level of resistance.

Data obtained from a thermal system capable of measuring changes in organ temperature as well as tissue thermal clearance in the uterus and vagina have been compared with blood flow measured continuously with a transit-time ultrasound volume-flow sensor placed around the common internal iliac artery and intermittently with radioactive microspheres in the chronically instrumented nonpregnant sheep. Temperature changes in both the uterus and the vagina correlated well with blood flow changes measured by both techniques after intravenous administration of estradiol or norepinephrine. Thermal clearance did not correlate well with blood flow in the vagina or uterus. These methods may have value in the investigation of blood flow patterns in various clinical situations such as the pelvic pain syndrome and early pregnancy.

The selective venoconstrictor dihydroergotamine (DHE) was given intravenously to 12 women with evidence of pelvic congestion. In 6 the effect of the drug on pelvic veins was observed by pelvic venography. After DHE there was a mean reduction of 35% in the diameter of the pelvic veins measured and the contrast medium cleared rapidly, with a visible reduction in pelvic congestion. In the other 6 women DHE was given during an acute attack of pelvic pain. The effect of the drug on pain relief was assessed by a single-blind crossover trial with intravenous saline as the placebo and by a visual analogue scale to assess the intensity of pain. Pain was significantly lower post-DHE 4 and 8 h and 2 and 4 days after treatment than after placebo. The results confirm a close association between demonstrable pelvic congestion and pelvic pain.

Counter-current chromatography is a form of liquid-liquid chromatography which uses low-speed centrifugation to hold one phase of an immiscible liquid pair stationary while the other is eluted through it. Two types of countercurrent chromatography are described: one suitable for preparative/analytical separation with aqueous-organic phase systems and the other for analytic fractionations using aqueous-aqueous phase systems. Applications of both processes are described, ranging from the purification of antibiotics, pesticides, and peptides to the fractionation of whole cells.