On the true identity of Sergentomyia gemmea and description of a closely related species: Se. raynali n. sp.

Several species of Leishmania are responsible for leishmaniases in Thailand, although little is known about their transmission. Sergentomyia gemmea has been suspected several times to transmit Leishmania martiniquensis. Some captures carried out in Thailand and Lao People's Democratic Republic have emphasized the scarcity of Se. gemmea, comprising only 1% of the collected females. The sequencing of cytochrome B mtDNA of our specimens showed that our specimens are not grouped with other Se. gemmea previously deposited in GenBank. The latter are grouped with some Se. khawi and Se. hivernus that we processed in the present study. We suspect misidentifications and propose focusing on the most useful characters for identification of Se. gemmea based on the examination of type-specimens. The examination of the ascoids exhibiting anterior spurs is the most important one. However, we also describe Se. raynali n. sp. exhibiting comparable spurs but differing from Se. gemmea by its original cibarium. Finally, the vectorial role of Se. gemmea appears very questionable in the absence of new evidence.

Recent developments in the management of anthelmintic resistance in small ruminants - an Australasian perspective.

There have been a number of significant advances in recent years to the theory and practice of managing anthelmintic resistance in sheep in Australasia. The general principles of resistance management are, firstly identification and mitigation of high-risk practices, secondly using effective anthelmintics, and thirdly maintaining a refuge of unselected parasites. The first of these principles has been updated recently with the findings from a series of farm-based trials in New Zealand, in which the economic benefits of both short- and long-acting anthelmintic treatments in ewes pre-lambing were found to be inconsistent and not always positive. There have also been significant changes to the second principle, particularly given the introduction of new active families onto the market. Evidence continues to favour the use of combination products to maximise efficacy and delay the onset of treatment-failure. Many farmers have readily accepted the effectiveness of maintaining a refuge of unselected parasites; the challenge for researchers and advisers is now to improve adoption of properly designed and implemented resistance management programmes. A recently completed education programme in New Zealand has demonstrated that when this is achieved, then anthelmintic resistance can be controlled, and in many cases reduced in severity.

Efferent intestinal lymph protein responses in nematode-resistant, -resilient and -susceptible lambs under challenge with Trichostrongylus colubriformis.

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 C3ß, C3α and haptoglobin (Hp) ß. In the R lambs C3α was significantly up regulated (P<0.05) on day 10, while in the Ri lambs Hp ß was significantly down regulated (P<0.05). In the S lambs, levels of C3ß were up regulated and levels of Hp ß 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.

The effect of purified condensed tannins of forage plants from Botswana on the free-living stages of gastrointestinal nematode parasites of livestock.

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.

The response of monocyte derived dendritic cells following exposure to a nematode larval carbohydrate antigen.

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.

Condensed tannins from Botswanan forage plants are effective priming agents of γδ T cells in ruminants.

The potential impact of extracts from forage plants on γδ T cell activity in ruminants was evaluated using an in vitro immunoassay. This study investigated whether plant extracts could prime γδ T cells via up-regulation of CD25 (interleukin-2 receptor alpha). Purified Sephadex LH-20 fractions, isolated from Viscum rotundifolium, Viscum verrucosum, Tapinanthus oleifolius and Grewia flava, were screened against γδ T cells on kid, lamb and calf peripheral blood lymphocytes. Condensed tannins (CT) from G. flava significantly primed γδ T cells in kids up to 64.75% at 10 µg/mL, which was statistically significant relative to the negative control at 22.66% (p=0.004). CT from T. oleifolius also induced priming of γδ T cells in kids, while fractions from V. rotundifolium and V. verrucosum induced minimal priming of γδ T cells. In contrast, there was no significant priming of γδ T cells from lambs and calves for any of the tested fractions (p>0.05). These findings suggest that CT from a selected range of Botswanan forage plants can stimulate the immune system in vivo in selected ruminant species and may participate in enhancing host innate immune responses.

Novel approaches for the control of helminth parasites of livestock VI: summary of discussions and conclusions.

Gastrointestinal helminth parasites impact on livestock production systems throughout the world, and the use of anthelmintics to control this problem has lead to the inevitable development of populations of helminths resistant to these treatments. This, coupled with consumer desires for minimal chemical inputs into food and fibre production, has prompted research into non-chemical approaches to helminth control. Scientists of the "Novel Approaches to the Control of Helminth Parasites of Livestock" group met for the 6th time in August 2010 and this paper summarises that meeting. Six scientific sessions addressed current approaches and topics of interest through formal presentations and discussion of issues raised by the contributing authors. Close interaction between researchers and extension specialists during the meeting has contributed to enhanced prospects for field application of research outcomes in the future.

Salivary IgA: a suitable measure of immunity to gastrointestinal nematodes in sheep.

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.

The production costs of anthelmintic resistance in sheep managed within a monthly preventive drench program.

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.

Protein separation using toroidal columns by type-J synchronous counter-current chromatography towards preparative separation.

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.

Samplers for evaluation and quantification of ultra-low volume space sprays.

A field study was conducted to explore the suitability of 5 pesticide deposition samplers for airborne spray and ground deposits from ultra-low-volume (ULV) space sprays. Samplers included horizontally stretched stationary cotton ribbons at 2 heights, rotating ribbon, rotating Teflon slides, and filter paper. Slides were also used for droplet-size analysis. A set of 7 samplers of each type was placed at 1, 7, 15, 25, 40, 65, and 90 m from the spray line along the spray swath. Water and BVA13 oil with fluorescent dyes as tracers were sprayed with the use of a truck-mounted ULV sprayer at dusk and dawn. Results suggest that the horizontal and rotating cotton ribbons are best for quantification of airborne spray and filter paper is best for ground deposition collection. The rotating slide samplers only detected the BVA13 oil-based sprays.

The use of phages for the removal of infectious biofilms.

Biofilm formation occurs spontaneously on both inert and living systems and is an important bacterial survival strategy. In humans biofilms are responsible for many pathologies, most of them associated with the use of medical devices. A major problem of biofilms is their inherent tolerance to host defences and antibiotic therapies; there is therefore an urgent need to develop alternative ways to prevent and control biofilm-associated clinical infections. Several in vitro experiments have shown that phages are able to infect biofilm cells and that those phages inducing the production of depolymerases have an advantage since they can penetrate the inner layers of the biofilm by degrading components of the biofilm exopolymeric matrix. In practice clinically relevant biofilms and especially those associated with the use of medical devices can possibly be controlled for example by the topic application or the impregnation of the surface of the device with a phage solution. Another interesting approach has been the use of a phage encoding a phage polysaccharide lyase to treat Pseudomonas aeruginosa biofilms in cystic fibrosis patients by aerosol administration. All these strategies require prior identification of the phage and/or polysaccharide depolymerase capable of infecting the bacterial cells and degrading the polysaccharide within the biofilm, respectively. The biofilm organisms must therefore be isolated and screened against a bank of phages. This procedure is essential and raises important biotechnological challenges: the existence of a bank of phages well characterised (physiologically and genetically) whose efficacy in vivo has been tested and pharmacokinetics studied; the existence of economical and safe production protocols and purification methods (e.g. the presence of endotoxins in a phage preparation may compromise phage therapy). It is however important to consider the fact that the chances of getting a specific phage with a high lytic capability and preferential expressing a relevant exopolymer degrading enzyme is likely to be low. Genetically engineered phages can play an important role in this process. Phages can be genetically manipulated to alter their host range and to induce the production of depolymerases. It is therefore important to reinforce the application of synthetic biology to engineer phages able to efficiently degrade medical biofilms. It is also important to develop efficient methods of phage delivery and to study "in vivo" the phage performance against biofilms. It is still not clear how effective the biofilm can be in protecting the phages against the immune system. Efficient and economic phage production and purification protocols need also to be addressed before one can hope to use phage treatment to prevent or control infectious biofilms.

Rapid linear scale-up of a protein separation by centrifugal partition chromatography.

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.

Multiple species of nematodes resistant to ivermectin and a benzimidazole-levamisole combination on a sheep farm in New Zealand.

AIM: To confirm the presence of multiple anthelmintic resistance on a sheep farm in New Zealand. METHODS: Three groups of 10 weaned Romney-cross lambs were treated either with an oral dose of ivermectin (0.2 mg/kg), or a benzimidazole/levamisole (BZ/LEV) combination (4.75 albendazole and 7.5 mg/kg levamisole), or were left untreated. Ten days later, animals were necropsied, and adult worms recovered and identified from the abomasa and small intestines. Pre- and post-treatment faecal nematode egg counts (FEC) were recorded, and larval cultures were performed. RESULTS: In a faecal egg count reduction test (FECRT), adjusted to reflect pre- and post-treatment larval culture results, ivermectin resistance was detected in Teladorsagia (Ostertagia), Trichostrongylus and Haemonchus spp, while BZ/LEV combination- resistant Teladorsagia and Trichostrongylus spp were also present. Adult worm counts confirmed these results, and identified the species involved as Teladorsagia circumcincta, Trichostrongylus colubriformis and H. contortus. CONCLUSION: Multiple, multi-generic anthelmintic resistance was confirmed on a sheep property in New Zealand. This included the first confirmed case of ivermectin resistance in T. colubriformis from sheep in New Zealand.

Intestinal amino acid absorption in lambs fed fresh Lucerne ( Medicago sativa) during an established Trichostrongylus colubriformis infection.

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.

Model for spiral columns and stationary phase retention in synchronous coil planet centrifuges.

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?"

Numerical model for the investigation of dual-flow in a spiral counter-current chromatography column.

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.

Spiral coils for counter-current chromatography using aqueous polymer two-phase systems.

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.

Seven-year follow-up of a randomized clinical trial comparing proton-pump inhibition with surgical therapy for reflux oesophagitis.

BACKGROUND: This randomized clinical trial compared long-term outcome after antireflux surgery with acid inhibition therapy in the treatment of chronic gastro-oesophageal reflux disease (GORD). METHODS: Patients with chronic GORD and oesophagitis verified at endoscopy were allocated to treatment with omeprazole (154 patients) or antireflux surgery (144). After 7 years of follow-up, 119 patients in the omeprazole arm and 99 who had antireflux surgery were available for evaluation. The primary outcome variable was the cumulative proportion of patients in whom treatment failed. Secondary objectives were evaluation of the treatment failure rate after dose adjustment of omeprazole, safety, and the frequency and severity of post-fundoplication complaints. RESULTS: The proportion of patients in whom treatment did not fail during the 7 years was significantly higher in the surgical than in the medical group (66.7 versus 46.7 per cent respectively; P=0.002). A smaller difference remained after dose adjustment in the omeprazole group (P=0.045). More patients in the surgical group complained of symptoms such as dysphagia, inability to belch or vomit, and rectal flatulence. These complaints were fairly stable throughout the study interval. The mean daily dose of omeprazole was 22.8, 24.1, 24.3 and 24.3 mg at 1, 3, 5 and 7 years respectively. CONCLUSION: Chronic GORD can be treated effectively by either antireflux surgery or omeprazole therapy. After 7 years, surgery was more effective in controlling overall disease symptoms, but specific post-fundoplication complaints remained a problem. There appeared to be no dose escalation of omeprazole with time.

Validation of candidate bovine reference genes for use with real-time PCR.

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.