Phenolic acid and flavonol water extracts of Delonix regia red flowers

Traditional healthy beverages in several African counties were home-made by water extraction of flowers of Delonix regia (Boj.) Raf. This tree belongs to the local biodiversity of plants used in the local medicine. It is also part of the traditional bioproducts marketed within African village communities. Scaling-up at pilot plant level, by mimicking this local recipe, lead to concentrated bioproducts containing various natural phenolic compounds, most of them have never been yet published. A three-steps process was used to prepare concentrated of water-extracted soluble phenolics. This process included: phenol extraction of dried plant by overnight diffusion in acidified water, clarification then concentration of the macerate, using membrane technology. Total phenol contents were determined as gallic acid equivalents using the Folin-Ciocalteu method. Total flavonoid contents, as delphinidin equivalents, were determined using the UV-vis colorimetric method. Comparison of concentration factors of both phenol families (phenolic acids and flavonols) contained in the 3 co-products obtained during the process were made between crude extracts, cross-flow microfiltration extracts and reverse osmosis concentrated extracts. Individual phenol compounds found in the products obtained were characterized and quantified by HPLC-DAD and by HPLC-MS². Among the 11 phenol compounds identified in the water-extracts, 3 have been already identified in the literature (2 phenolic acids and 1 flavonol), and 8 new compounds, in noticeable amounts, have never been cited. We have confirmed the presence of phenolic acids such as 3,4,5-trihydroxybenzoic (gallic acid), 3,4-dihydroxybenzoic (protocatechuic acid) and of quercetin. We have characterized for the first time the presence of 2-hydroxy 5-[(3,4,5 trihydroxyphenyl) carbonyl oxy] benzoic acid and of 7 other flavonols belonging to the quercetin, kaempferol and isorhamnetol compound families. Using both LCMS and NMR analyses, they have been identified and quantified as: rutin, quercetin 3-O-glucoside, quercetin 3-O-galactoside, quercetin trihexoside, quercetin 3-O-robinobioside, kaempferol rhamnosylhexoside and isorhamnetol rhamnosyl-hexoside.     

Development of Highly Selective Kv1.3-Blocking Peptides Based on the Sea Anemone Peptide ShK

ShK, from the sea anemone Stichodactyla helianthus, is a 35-residue disulfide-rich peptide that blocks the voltage-gated potassium channel Kv1.3 at ca. 10 pM and the related channel Kv1.1 at ca. 16 pM. We developed an analog of this peptide, ShK-186, which is currently in Phase 1b-2a clinical trials for the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. While ShK-186 displays a >100-fold improvement in selectivity for Kv1.3 over Kv1.1 compared with ShK, there is considerable interest in developing peptides with an even greater selectivity ratio. In this report, we describe several variants of ShK that incorporate p-phophono-phenylalanine at the N-terminus coupled with internal substitutions at Gln16 and Met21. In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus. Their selectivity was determined by patch-clamp electrophysiology on Kv1.3 and Kv1.1 channels stably expressed in mouse fibroblasts. The peptides with an alanine extension blocked Kv1.3 at low pM concentrations and exhibited up to 2250-fold selectivity for Kv1.3 over Kv1.1. Analogs that incorporates p-phosphono-phenylalanine at the N-terminus blocked Kv1.3 with IC₅₀s in the low pM range and did not affect Kv1.1 at concentrations up to 100 nM, displaying a selectivity enhancement of >10,000-fold for Kv1.3 over Kv1.1. Other potentially important Kv channels such as Kv1.4 and Kv1.6 were only partially blocked at 100 nM concentrations of each of the ShK analogs.     

First detection of "Candidatus Mycoplasma haemolamae" in South American Camelids of Switzerland and evaluation of prevalence

Haemotrophic mycoplasmas (also known as haemoplasmas), small bacterias which parasite the surface of erythrocytes, have been described in several species. Recently, molecular methods were developed for the diagnosis of haemoplasma infection. The presented study describes the first detection and the investigation of prevalence of "Candidatus Mycoplasma haemolamae" in South American Camelids in Switzerland. A random sample of the latter population was tested for haemoplasma infections using real-time PCR. The infection was detected in 18.6% of the animals and was found both in indigenous and in imported camelids. Of the tested herds 39,1% harboured at least one animal positive for haemoplasmas in PCR. There was no difference in prevalence between male and female animals and llamas and alpacas, respectively. Furthermore, the prevalence of infection was not significantly different in diseased animals compared to healthy camelids. From the latter observation and the fact that the high prevalence was accompanied by an undetectable incidence, we concluded that the pathogenicity of "Candidatus Mycoplasma haemolamae" may be low.     

Effects of medetomidine-midazolam-fentanyl IV bolus injections and its reversal by specific antagonists on cardiovascular function in rabbits

The objective of this study was to investigate the short-term cardiovascular effects of intravenous (IV) medetomidine-midazolam-fentanyl (MMF) injections in the rabbit using vascular ultrasonography and echocardiography.Anesthesia with MMF was induced intramuscularly (IM) in 8 female New Zealand White rabbits before 3 defined bolus injections of MMF were given IV. Before and for 10 min after each MMF injection the following vascular variables [at the left common carotid artery (ACC) after the first injection and at the abdominal aorta (AA) after the second injection]: vessel diameter (D), peak systolic, minimum diastolic, end-diastolic and average blood flow velocities (psBFV, mdBFV, edBFV, Vave), average volumetric flow (VFave), resistance index (RI) and pulsatility index (PI) and other clinical variables: mean arterial pressure (MAP), heart rate (HR), peripheral arterial oxygen saturation and end-tidal CO₂ were recorded. Echocardiography was used after the third injection to investigate changes in cardiac parameters. Additionally, hemodynamic effects were observed at the ACC after complete subcutaneous antagonism of anesthesia by atipamezole-flumazenil-naloxone (AFN) until recovery of the animals.Medetomidine-midazolam-fentanyl IV caused a significant decrease of blood flow velocity in both investigated vessels which was associated with a significant decrease of HR and cardiac performance indicated by the decrease of FS and average volumetric blood flow. Mean arterial pressure significantly increased after each MMF injection; whereas, it significantly decreased after AFN injection. Therefore, MMF and AFN should be carefully used in rabbits and may not be suitable in patients with ventricular dysfunction.     

Dynamics of serum antibodies to and load of porcine circovirus type 2 (PCV2) in pigs in three finishing herds,affected or not by postweaning multisystemic wasting syndrome

Background

Despite that PMWS commonly affects pigs aged eight to sixteen weeks; most studies of PMWS have been conducted during the period before transfer to finishing herds. This study focused on PCV2 load and antibody dynamics in finishing herds with different PMWS status.

Methods

Sequentially collected blood samples from 40 pigs in each of two Swedish (A and B) and one Norwegian (C) finishing herds were analysed for serum PCV2-load and -antibodies and saliva cortisol. The two Swedish herds differed in PMWS status, despite receiving animals from the same sow pool (multi-site production). However, the PMWS-deemed herd (A) had previously also received pigs from the spot market. ResultsThe initial serum PCV2 load was similar in the two Swedish herds. In herd A, it peaked after two weeks in the finishing herd and a high number of the pigs had serum PCV2 levels above 10⁷ per ml. The antibody titres increased continually with exception for the pigs that developed PMWS, that had initially low and then declining antibody levels. Pigs in the healthy herd B also expressed high titres of antibodies to PCV2 on arrival but remained at that level throughout the study whereas the viral load steadily decreased. No PCV2 antibodies and only low amounts of PCV2 DNA were detected in serum collected during the first five weeks in the PMWS-free herd C. Thereafter a peak in serum PCV2 load accompanied by an antibody response was recorded. PCV2 from the two Swedish herds grouped into genotype PCV2b whereas the Norwegian isolate grouped into PCV2a. Cortisol levels were lower in herd C than in herds A and B.

Conclusions

The most obvious difference between the Swedish finishing herds and the Norwegian herd was the time of infection with PCV2 in relation to the time of allocation, as well as the genotype of PCV2. Clinical PMWS was preceded by low levels of serum antibodies and a high load of PCV2 but did not develop in all such animals. It is notable that herd A became affected by PMWS after errors in management routine, emphasising the importance of proper hygiene and general disease-preventing measures.     

Seasonal influences on livestock keeping in a sedentary crop–livestock system

Although seasonality is recognised as an important part of agricultural-based livelihoods, limited literature is available on the subject area, and it is often only alluded to in discussion of other aspects of rural livelihoods. A 2-year longitudinal study to examine the influences of seasonal changes on livestock keeping in a smallholder crop–livestock production system was carried out in Busia District, western Kenya. The study aimed to obtain a picture of yearly variations in household activities and resources, as a means of understanding decisions made by households regarding animal health management, and household times of vulnerability in terms of losing animals. Data collection coincided with the three main seasons in the study area. Information on (inter alia) seasonal livelihood activities, animal health care expenses, numbers of disease episodes and livestock movements into and out of households was collected using questionnaires and participatory rural appraisal methodologies. Farmers suggested clear and consistent seasonal changes and events, but data analyses did not show the patterns expected in relation to livestock keeping. Important observations were made in relation to livestock disease episodes and the use of veterinary services; livestock disease episodes were higher during the long rains than in the dry season, but more money was spent during the dry season when numbers of disease episodes were low, and more households also used professional veterinary services during this season (χ ² = 81.47, P < 0.001). In both study years, a higher proportion of households treated animals themselves during the rainy seasons (z = −2.4, P = 0.02; z = −5.03, P < 0.001).     

Development and application of a real-time TaqMan(®) qPCR assay for detection and quantification of 'Candidatus Mycoplasma haemolamae' in South American camelids

Two alpacas from a herd in southwest Switzerland died for unknown reasons. Necropsy revealed chronic weight loss and pale mucous membranes. Infection with hemotropic mycoplasmas was suspected and subsequently confirmed by molecular methods. In order to investigate the epidemiological situation in this herd, a real-time TaqMan(?) qPCR assay for the specific detection and quantification of hemoplasma infection in South American camelids was developed. This assay was based on the 16S rRNA gene and amplified 'Candidatus Mycoplasma haemolamae' DNA, but not DNA from other hemoplasmas or non-hemotropic mycoplasma species. The lower detection limit was one copy/PCR, and the amplification efficiency was 97.4%. In 11 out of 24 clinically healthy herd mates of the two infected alpacas, 'Candidatus M. haemolamae' infection was confirmed. No correlation was found between bacterial load and clinical signs or anemia. The assay described herein enables to detect and quantify 'Candidatus M. haemolamae' and may be used in future studies to investigate the prevalence, pathogenesis and treatment follow-up of hemoplasma infections in South American camelids.