Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale.

l-Ascorbic acid (vitamin C) supplementation to optimize health and reproduction in cattle.

Cattle can synthesize L-ascorbic acid (or Vitamin C) from either D-glucose or D-galactose through glucuronic acid pathway in the liver. L-Ascorbic acid present in cattle diet is almost totally destroyed by rumen microorganisms making them essentially dependent on its endogenous synthesis, which is assumed sufficient to meet the physiological requirement. Therefore, the role of vitamin C in cattle health and disease has remained widely overlooked. However, there is mounting evidence that the level of L-ascorbic acid in blood and other tissues decreases in association with stress and disease, and Vitamin C supplementation revealed favorable response as evident from early recovery. The present review is an attempt to summarize the existing literature pertaining to the physiological role of L-ascorbic acid and the scope of its supplementation in the prevention and treatment of diseases in cattle. It should be realized that the aqueous solution of vitamin C is highly acidic and subcutaneous or intramuscular administration may cause tissue irritation and inflammation, whereas the sodium ascorbate solution is less acidic and might be used for intramuscular administration.

Effect of mitotic inducers and retinoic acid blocker on expression of pluripotent genes in ES cells derived from early stage in vitro-produced embryos in buffalo.

So far, it has been difficult to generate embryonic stem (ES) cell from early stage preimplantation embryos of buffalo. These ES cells will be more helpful for efficient embryo cloning and generation of body cells as they are more primitive than inner cell mass (ICM)-derived ES cells. The present study was conducted to find the effect of lipopolysaccharide (LPS), melatonin (N-acetyl-5-methoxytryptamine, a pineal gland product), and citral (3,7-dimethyl-2,6-octadienal and a retinoic acid synthesis blocker) on establishment of primary ES cell colonies, the comparative size of the ES cell colonies, and expression of pluripotent genes during extended period of culture in buffalo. Zona-free eight-cell stage in vitro fertilization (IVF) embryos were cultured in ES cell medium supplemented with none (media I as control), LPS (media II), citral melatonin (media III), or melatonin (media IV). The multiplication of blastomere leading to ES cell colony formation and expression of pluripotent genes were assessed up to day 20 of culture. The primary colony formation, the comparative size of the ES cell colonies, and expression of pluripotent genes in these colonies were better in the medium supplemented with melatonin in all days of culture. Within melatonin supplementation, the colony size was comparatively larger on day 8 and day 12 of culture. Further, with this supplementation, the Oct-4 and Nanog expression was comparatively higher on all days of culture. The results indicated that supplementation of melatonin helped in the formation of better primary ES cell colony as well as in the maintenance of pluripotency. The results also indicated that primary colonies developed on day 8 to day 12 of culture may be better for passaging them for establishment of ES cell line from early stage preimplantation IVF embryos of in buffalo.

Ascorbic acid, garlic extract and taurine alleviate cadmium-induced oxidative stress in freshwater catfish (Clarias batrachus).

An experiment was conducted to investigate bioaccumulation potential of cadmium (Cd) and changes in oxidative stress indices in liver and kidney tissues from Cd-exposed catfish (Clarias batrachus) with or without simultaneous treatment of water with ascorbic acid, garlic extract or taurine. C. batrachus (n=324) with average length of 20+/-4 cm and weight of 86+/-5 g were used for the present investigation. Fishes were divided into nine groups (I to IX) each comprising 36 fishes. The fishes of groups II, III, IV and V were challenged with 5 ppm of cadmium chloride monohydrate (CdCl2.H2O), whereas groups VI, VII, VIII and IX were exposed to 10 ppm CdCl2.H2O solution for a period of 45 days. Group I was kept as negative control and the fishes of this group were maintained in water containing no added Cadmium. Group II and VI were maintained as Cd exposed non treated control to serve as positive controls. Fishes of III and VII, IV and VIII, V and IX received ascorbic acid (5 ppm), extract of dried garlic (5 ppm) or taurine (5 ppm), respectively during the entire experiment period. The concentrations of Cd in liver and kidney increased significantly following exposure to Cd and the level continued to rise with the increase in exposure duration. Treatment of tank water with ascorbic acid, garlic or taurine significantly reduced the Cd concentrations in tissues compared to the positive control group, but the level in Cd exposed groups was greater than the negative control group. Fishes exposed to Cd and treated with ascorbic acid, garlic or taurine had reduced oxidative stress as evidenced from lower concentration of lipid peroxides and higher activities of superoxide dismutase and catalase in liver, kidney and erythrocytes compared to fishes exposed to Cd. The reduction in Cd induced oxidative stress was highest in ascorbic acid treated group followed by garlic and taurine treatment. The results suggest that ascorbic acid, garlic and taurine have potential to reduce tissue accumulation of Cd and associated oxidative stress in freshwater catfish.

Tamarindus indica L. and Moringa oleifera M. extract administration ameliorates fluoride toxicity in rabbits.

Aqueous extracts of T. indica fruit pulp (100 mg/kg body weight) and M. oleifera seeds (50 mg/kg body wight) orally once daily for 90 days lowered plasma fluoride concentrations in rabbits receiving fluorinated drinking water (200 mg NaF/ Liter water). Cortical indices and metaphysial width in animals receiving extracts also revealed beneficial effects of plant extracts. Changes in plasma biochemistry suggested less hepatic and renal damages in animals receiving plant extracts along with fluorinated water in comparison to that receiving fluorinated water alone. Preliminary results revealed these plant extracts have some potential to mitigate fluoride toxicity.

Changes in oxidative stress indices, zinc and copper concentrations in blood in canine demodicosis.

Status of certain oxidative stress indices and zinc and copper concentrations in blood were estimated in dogs with localized demodicosis (LD) and generalized demodicosis (GD). In comparison to healthy control, erythrocytic lipid peroxides level and superoxide dismutase activity were significantly (P<0.01) higher in both LD as well as GD. However, level of reduced glutathione and activity of catalase were significantly (P<0.01) lower in both LD and GD. Blood zinc and copper levels in dogs with LD and GD were significantly (P<0.01) lower than healthy control. Significant (P<0.01) differences were also observed in different oxidative stress indices and zinc and copper levels in between LD and GD groups. From the present study, it was concluded that demodicosis is associated with oxidative stress and antioxidant supplementation may be beneficial in management of canine demodicosis.

Enhanced erythrocytic lipid peroxides level in rabbits after repeated parental administration of iron.

An experiment was conducted in rabbits to evaluate the possible involvement of oxidative stress in iron-overload animals. Ten adult female New Zealand white rabbits were divided into 2 equal groups with 5 animals each. Group II animals received intramuscular iron dextran injections (120 mg/kg body wt/day) on alternate day for 14 days (8 injections), while Group I animals did not receive any iron supplementation to serve as negative controls. The blood samples were collected by cardiac puncture before the start of iron dosing and thereafter, at weekly intervals for 28 days. The samples were processed to measure blood iron concentration, packed cell volume, erythrocytic lipid peroxide (LPO) level, superoxide dismutase (SOD) and catalase (CAT) activities. The blood iron concentration showed a rising trend following repeated iron administration, and the mean level recorded on day 14 was significantly higher than respective day 0 value. LPO level remained significantly higher from day 14 onwards till the end of the observation period of 14 more days after cessation of iron adminstration. Erythrocytic superoxide dismutase activities showed a transient significant rise on day 7, and thereafter, showed a declining trend, but remained statistically comparable to respective day 0 or corresponding value of the control animals.

Acupressure for prevention of pre-operative anxiety: a prospective, randomised, placebo controlled study.

Pre-operative anxiety is associated with many unwanted effects such as increased analgesic and anaesthetic requirement, postoperative pain and prolonged hospital stay. In the present study, we investigated the effects of acupressure on pre-operative anxiety and bispectral index (BIS) values. Seventy-six adults, ASA grade I and II, undergoing elective surgery, were randomly assigned to two equal groups. Group 1 (control) received acupressure at an inappropriate site and group 2 (acupressure) received acupressure at extra 1 point. The study was conducted during the pre-operative period and the duration of the study was 40 min (acupressure was applied for 10 min and thereafter patients were observed for another 30 min). Anxiety was recorded on a visual stress scale (VSS) at the start of the study and thereafter at 10 and 40 min. BIS was recorded at 0, 2, 5, 10, 12, 15, 30 and 40 min. The VSS decreased in both groups following pressure application for 10 min: median VSS (interquartile range) were 5 (1) vs. 8 (1) in the acupressure and 7 (0) vs. 8 (1) in the control groups (p < 0.001). Both pre-operative anxiety and BIS decreased significantly during acupressure application at extra 1 point (p < 0.001). Acupressure is effective in decreasing both pre-operative anxiety and BIS; however, these effects are not sustained 30 min following release of acupressure. Further studies are needed to elucidate the duration for which acupressure is effective.

Enhanced erythrocytic lipid peroxides and reduced plasma ascorbic acid, and alteration in blood trace elements level in dairy cows with mastitis.

Oxidative stress has been associated in several inflammatory conditions and incriminated in the pathogenesis of many diseases. However, little information is available on the status of plasma antioxidant levels, essential components of important antioxidant enzymes such as copper, zinc and selenium in blood, and the end product of oxidative damage to the erythrocytic polyunsaturated fatty acids in inflammatory udder conditions. Blood samples were collected from three groups of dairy cows, with 21 in each group: animals with healthy udder, clinical mastitis, and subclinical mastitis. These animals were randomly selected from a herd on the basis of the California mastitis test, somatic cell count and total bacterial count. The mean plasma ascorbic acid concentration was significantly lower in cows with subclinical (p = 0.004) and clinical mastitis (p = 0.000) and the erythrocytic lipid peroxide levels were significantly (p = 0.000) higher in clinical mastitis as compared to controls. There was a significant decrease in mean blood zinc concentration in subclinical (p = 0.005) and clinical mastitis (p = 0.000), but an increase in mean blood copper level in the clinical mastitis group. It was concluded that the blood antioxidant status declines in inflammatory udder conditions, suggesting that incorporation of antioxidants may help in better management of mastitis in dairy cows.

Cysteine string protein is required for calcium secretion coupling of evoked neurotransmission in drosophila but not for vesicle recycling.

The entire deletion of the cysteine string protein (CSP) gene causes a temperature-sensitive (ts) block of evoked neurotransmission in Drosophila. CSP has been found to interact in vitro with the clathrin-uncoating ATPase HSC70, suggesting a potential role of CSP in vesicle recycling. Using FM1-43 imaging, we analyzed whether the ts block of neurotransmission in csp mutants is caused by a defect in vesicle exocytosis or vesicle recycling. We determined that FM1-43-labeled synaptic boutons of csp mutant neuromuscular junctions fail to destain at 32 degrees C after K+ depolarization, and that FM1-43 dye uptake cannot be evoked by K+ stimulation at 32 degrees C. However, when we stimulated dye uptake independent of depolarization by using black widow spider venom (BWSV), we observed endocytotic uptake of FM1-43. This suggests that endocytosis exhibits no primary ts defect. In addition, we found no ts defect of vesicle recycling at 32 degrees C that would correlate with the ts block of neurotransmission. We also discovered that BWSV and the calcium ionophore calcimycin stimulate FM1-43 destaining and quantal release in csp mutants at 32 degrees C when depolarization fails to evoke any response. The wild-type-like, calcimycin-induced response in csp null mutants indicates that some aspect of the depolarization-dependent calcium signaling pathway must be impaired, either calcium entry, calcium action, or both. Collectively, our results indicate that the csp mutation affects calcium secretion coupling of evoked exocytosis but not vesicle recycling. This supports the hypothesis that CSP links synaptic vesicles to calcium secretion coupling.

Increased serum interleukin-1-receptor-antagonist concentrations in major depression.

Recently, it has been shown that major depression may be accompanied by an increased production of interleukin-1 beta (IL-1 beta), an acute phase (AP) response and simultaneous signs of activation and suppression of cell-mediated immunity. The interleukin-1-receptor antagonist (IL-1-rA) is released in vivo during an AP response and serum levels are increased in many immune disorders. The release of IL-1-rA may limit the pro-inflammatory effects of IL-1. This study has been carried out to examine serum IL-1-Ra in 68 depressed subjects (21 minor, 25 simple major and 22 melancholic subjects) vs. 22 normal controls. Depressed subjects showed significantly higher serum IL-1-rA concentrations than healthy controls. 29% of all depressed subjects had serum IL-1-rA levels higher than the mean value +2 standard deviations of normal controls; 44% depressed subjects had IL-1-rA values greater than 0.215 ng/ml with a specificity of 90%. In depressed subjects, there was a significant and positive relationship between serum IL-1-rA and severity of illness. In depression, there were no significant relationships between serum IL-1-rA concentrations and indicants of hypothalamic-pituitary-adrenal (HPA)-axis activity, such as 24-h urinary cortisol and postdexamethasone cortisol values. Women had significantly higher serum IL-1-rA levels than men. The findings support the thesis that depression is accompanied by an immune-inflammatory response.

Increased plasma concentrations of interleukin-6, soluble interleukin-6, soluble interleukin-2 and transferrin receptor in major depression.

Recently, it was found that major depression may be accompanied by significant changes in cell-mediated and humoral immunity. The purpose of this study was to investigate the plasma concentrations of interleukin (IL)-6, soluble IL-6 receptor (sIL-6R), sIL-2R and transferrin receptor (TfR) in patients with major depression in an acute phase of illness, in remission and during antidepressive treatment. Plasma concentrations of IL-6, sIL-6R, sIL-2R and TfR were significantly higher in major depressed subjects than in healthy controls. In major depressed subjects, but not in normal controls, there were significant positive correlations between the plasma concentrations of IL-6 and sIL-6R, IL-6 and sIL-2R, IL-6 and TfR, and between sIL-2R and TfR. Subchronic treatment with antidepressive drugs, such as fluoxetine or tricyclic antidepressants, did not significantly affect plasma IL-6, sIL-6R, sIL-2R or TfR. The latter did not significantly differ between major depressed patients in an acute phase of illness or in complete clinical remission. It is suggested that: (1) a coordinated and upregulated production of IL-6, sIL-6R, sIL-2R and TfR may constitute a trait marker of major depression; and that (2) an upregulated production of IL-6 may represent a contributing factor to the various immune disorders encountered in major depression and maybe to the pathophysiology or pathogenesis of that illness.