A rapid and simple method for the separation of pure lymphocytes from horse blood

A method for the separation of pure and viable lymphocytes and granulocytes from the same blood sample in horses was reported. By centrifuging equine heparinized blood at 100 xg for 10 min at room temperature (r.t.), the resulting supernatant plasma was an almost pure (97.71 +/- 0.30%; n = 15) suspension of highly viable (98.72 +/- 0.28%) lymphocytes. When sodium citrate was used as an anticoagulant, lymphocyte suspensions collected in the same manner showed lower purity (87.89 +/- 1.59%; n = 9) and higher yields (56.56 +/- 3.89%, n = 9 versus 36.11 +/- 2.23%, n = 15). Where needed, a further centrifugation at 250 xg for 3 min (r.t.) of heparinized lymphocyte preparations removed an average of 87.39% (n = 15) contaminating platelets. A suspension of 85.96 +/- 2.20% pure granulocytes (93.23 +/- 1.74% neutrophils; n = 14) with minimal contamination by erythrocytes and high viability (93.11 +/- 1.26%) was obtained by performing a flash red blood cell lysis on the white-greyish layer resulting from the centrifugation of the heparinized blood samples. Among the several methods available, the procedure described herein is easy, rapid, cheap and reproducible.     

Effects of three esca-associated fungi on Vitis vinifera L.: III. Enzymes produced by the pathogens and their role in fungus-to-plant or in fungus-to-fungus interactions

When the esca-associated fungi Phaeomoniella chlamydospora (Pch), Togninia minima (Tmi) and Fomitiporia mediterranea (Fme) were grown in liquid stationary cultures, it was seen that they were able to live in media containing resveratrol (RES) or tannic acid (TA) as the sole carbon source and that the fungi were able to convert both compounds. Particular attention is paid here to detecting RES and TA conversion. Pch, Tmi and Fme were partially inhibited by RES or TA. Pch, Tmi and Fme produced extracellular tannase, laccase and peroxidase enzymes in liquid or agarized cultures, whether glucose was present or not. When colonies of Pch, Tmi and Fme were confronted, they showed spatially and temporally heterogeneous patterns of laccase and peroxidase activity. The results indicate the non-synergistic, competitive association of Pch and Tmi and the inhibition of Fme growth. Muconic acid, a well-known intermediate in a large number of lignin and phenol oxidative processes, can partly or completely inhibit the lignolytic agent Fme, but is tolerated by Pch and Tmi. An explanation for wood pigmentation patterns by Pch, Tmi and Fme is given.     

Effects of three esca-associated fungi on Vitis vinifera L.: V. Changes in the chemical and biological profile of xylem sap from diseased cv. Sangiovese vines

A vineyard of Vitis vinifera cv. Sangiovese was surveyed for incidence of esca and xylem sap collection. Sap samples were collected from healthy vines and from those with dual infection by Phaeomoniella chlamydospora (Pch) and Togninia minima (Tmi) or triple infection by Pch, Tmi and Fomitiporia mediterranea (Fme), during each early spring in a 3-year period (2001–2003). In order to analyse the possible trends in the climatic data, temperature and rainfall were assessed. At sap harvesting, aliquots of sap were assayed for phytotoxicity and extracted with ethyl acetate for phytotoxin recovery. Moreover, the exopolysaccharide (EPS) content was evaluated on several sap samples during the bleeding period. Conidia of Pch and Tmi, mycelium of Fme and their secondary metabolites were found in the sap of the esca-affected vines, indicating that the pathogens and their by-products together with some defence substances were accumulated and then translocated. Bioactivity tests showed toxicity of the sap from esca-affected vines to healthy detached leaves of cv. Sangiovese. The daily amount of sap, the pH, and the volume (J_v) and solute (J_s) fluxes were analysed as a function of the infecting fungi. Pullulan, glucogalactomannan(s) and arabinogalactan(s) are the main EPS in the esca-infected vines, whereas in the sap of healthy vines no traces of pullulan were found. Scytalone and isosclerone usually produced in vitro by Pch and Tmi were also detected in the sap of vines infected by Pch and Tmi or by Pch, Tmi and Fme. The endogenous phytohormone content of healthy vines evaluated by the cutting bioassay was different from that of infected vines. Four phenolics belonging to three classes e.g., benzoic acid derivatives, stilbenes and flavonol-glycosides were separated and identified by HPLC.     

Synthesis and in-vitro antifungal activity of 6-trifluoromethyipyrazolo[3,4-6d]pyrimidin-4(5H)-thiones

Twelve 6-trifluoromethylpyrazolo[3,4-d]pyrimidin-4(5H)-thiones were prepared by the reaction between 4-thiocarbamoyl-5-aminopyrazoles and trifluoroacetic anhydride. They were tested in vitro for antifungal activity against a series of phytopathogenic fungi of different taxonomic classes. The EC50 and MIC values of four compounds were comparable or inferior to those of reference commercial fungicides in controlling Sclerotinia minor, Corticium solani and Phytium ultimum.     

Infection, immunity and the neuroendocrine response

The Central Nervous (CNS) and Immune Systems (IS) are the two major adaptive systems which respond rapidly to numerous challenges that are able to compromise health. The defensive response strictly linking innate to acquired immunity, works continuously to limit pathogen invasion and damage. The efficiency of the innate response is crucial for survival and for an optimum priming of acquired immunity. During infection, the immune response is modulated by an integrated neuro-immune network which potentiates innate immunity, controls potential harmful effects and also addresses metabolic and nutritional modifications supporting immune function. In the last decade much knowledge has been gained on the molecular signals that orchestrate this integrated adaptive response, with focus on the systemic mediators which have a crucial role in driving and controlling an efficient protective response. These mediators are also able to signal alterations and control pathway dysfunctions which may be involved in the persistence and/or overexpression of inflammation that may lead to tissue damage and to a negative metabolic impact, causing retarded growth.This review aims to describe some important signalling pathways which drive bidirectional communication between the Immune and Nervous Systems during infection. Particular emphasis is placed on pro-inflammatory cytokines, immunomodulator hormones such as Glucocorticoids (GCs), Growth hormone (GH), Insulin-like Growth Factor-1 (IGF-1), and Leptin, as well as nutritional factors such as Zinc (Zn).Finally, the review includes up-to-date information on this neuroimmune cross-talk in domestic animals. Data in domestic animal species are still limited, but there are several exciting areas of research, like the potential interaction pathways between mediators (i.e. cytokine-HPA regulation, IL-6-GCS-Zn, cytokines-GH/IGF-1, IL-6-GH-Leptin and thymus activity) that are or could be promising topics of future research in veterinary medicine.     

Long‐term residence of juvenile loggerhead turtles to foraging grounds: a potential conservation hotspot in the Mediterranean

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Serological and parasitological survey of dourine in the Arsi–Bale highlands of Ethiopia

This study was conducted from August 2005 to January 2007 to determine prevalence and distribution of dourine in horses and to investigate the occurrence of clinical and carrier cases in donkeys and mules in the Arsi–Bale highlands. Study methodology was based on questionnaire, serological, clinical and parasitological survey. The questionnaire indicated that dourine is a major health problem of equines in the Arsi–Bale highlands. Though dourine is commonly observed throughout the year, it has a seasonal character and occurs mostly during the breeding season from June to late September. Serological screening of 646 horses showed a seroprevalence of 184 (28%), 161 (25%) and 125 (19%) for card agglutination test for trypanosomosis, LATEX and enzyme-linked immunosorbent assay, respectively. Risk factors were parity number, previous history of abortion and body condition score. No trypanosomes could be detected by Giemsa staining or by haematocrit centrifugation technique. Ten puppies inoculated with blood samples, genital washes and oedematous fluids remained parasitologically negative. Different characteristic signs of dourine were observed. During the genital stage, mares showed vaginal oedema, discharge and presence of depigmented scars over the external genitalia. In stallions, oedema of the scrotum and prepuce, prepucial and urethral discharge, and ulceration of the genital mucosae mainly of the penile were observed. In both sexes, lameness in one or both legs, partial dragging and stiffness of the hind legs and incoordination were the dominant signs observed as nervous form of the disease.     

Long-term Electrocardiography Recording with Holter Monitoring in 15 Donkeys

The heart rates (HR) and rhythms of 15 healthy donkeys of various ages, both sexes, and various breeds were analyzed throughout 24-hour Holter monitoring. The animals were evaluated at rest in their daily environment without the presence of investigators causing stress, using a three-channel digital Holter recorder. Analysis revealed a maximal HR range from 62.5 to 93.7 beats/min (mean, 72.50 ± 7.51 beats/min), whereas the minimal HR ranged from 29.7 to 42.2 beats/min (mean, 34.90 ± 4.22 beats/min). The daily mean HR was 47.55 ± 3.70 beats/min. The daytime mean HR was 49.39 ± 2.77 beats/min, whereas the night time rate was 46.22 ± 4.38 beats/min. Sinus bradycardia and sinus tachycardia were detected in 7 and 10 of 15 studied donkeys, respectively. Cardiac dysrhythmias due to high vagal tone such as sinoatrial heart block and second-degree atrioventricular heart block were occasionally recorded in 1 and 3 donkeys, respectively. A higher mean HR and fewer cardiac dysrhythmias were observed in donkeys than in horses and ponies at rest. These findings could be justified by differences in the autonomic nervous system tone.     

Yield response of corn to irrigation and nitrogen fertilization in a Mediterranean environment

Productivity and resource-use efficiency in corn (Zea mays L.) are crucial issues in sustainable agriculture, especially in high-demand resource crops such as corn. The aims of this research were to compare irrigation scheduling and nitrogen fertilization rates in corn, evaluating yield, water (WUE), irrigation water (IRRWUE) and nitrogen use (NUE) efficiencies. A 2-year field experiment was carried out in a Mediterranean coastal area of Central Italy (175 mm of rainfall in the corn-growing period) and corn was subjected to three irrigation levels (rainfed and supply at 50 and 100% of crop evapotranspiration, ETc) in interaction with three nitrogen fertilization levels (not fertilized, 15 and 30 g (N) m⁻²). The results indicated a large yearly variability, mainly due to a rainfall event at the silking stage in the first year; a significant irrigation effect was observed for all the variables under study, except for plant population. Nitrogen rates affected grain yield plant⁻¹ and ear⁻¹, grain and biomass yield, HI, WUE, IRRWUE and NUE, with significant differences between non-fertilized and the two fertilized treatments (15 and 30 g (N) m⁻²). Furthermore, deficit irrigation (50% of ETc) was to a large degree equal to 100% of the ETc irrigation regime. A significant interaction “N × I” was observed for grain yield and WUE. The effect of nitrogen availability was amplified at the maximum irrigation water regime. The relationships between grain yield and evapotranspiration showed basal ET, the amount necessary to start producing grain, of about 63 mm in the first and 206 mm in the second year. Rainfed crop depleted most of the water in the 0–0.6 m soil depth range, while irrigated scenarios absorbed soil water within the profile to a depth of 1.0 m. Corn in a Mediterranean area can be cultivated with acceptable yields while saving irrigation water and reducing nitrogen supply and also exploiting the positive interaction between these two factors, so maximizing resource-use efficiency.