NOPR Collection: [Pages 215-280]

Cloning and expression of cultural filtrate proteins from novel and native strains of Mycobacterium avium subspecies paratuberculosis and their application in ELISA based sero-diagnosis of Johne's disease

2021-04-01T00:00:00Z

Title: Cloning and expression of cultural filtrate proteins from novel and native strains of Mycobacterium avium subspecies paratuberculosis and their application in ELISA based sero-diagnosis of Johne's disease Authors: Chaubey, Kundan Kumar; Gupta, Rinkoo Devi; Singh, Shoor Vir; Bhatia, Ashok Kumar; Gupta, Saurabh; Kumar, Naveen; Rathore, Abhishek Singh; Singh, Manju; Stephen, Bjorn John; Dhama, Kuldeep Abstract: Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is endemic in livestock leading to low per animal productivity. MAP as survives pasteurization, poses a public health problem because of high exposure to animals and humans. There is an urgent need for newer diagnostic tests with high specificity and sensitivity as the current ones suffer from lower sensitivity and specificity. In present study, six Mycobacterium avium subspecies paratuberculosis (MAP)-specific culture filtrate proteins (CFPs) were produced and evaluated for sero-diagnosis of MAP infection in goat and cattle herds in India. Genes encoding for six MAP-CFPs were amplified and cloned into easy cloning vector pJET1.2/pTZ57R followed by sub-cloning into expression vector pET28a (+)/pET22b (+) containing C-terminal Histidine. Recombinant CFPs (r-CFPs) expressions were optimized in Escherichia coli (Rosetta cells) and purified using Ni-NTA affinity chromatography. In SDS-PAGE, MAP CFPs viz., 1693c, 2168c, ModD, 85C, Pep AN and Pep AC showed 22, 24, 55, 38, 20 and 25 kDa molecular masses, respectively. Identity of these r-CFPs was further confirmed by immuno-blotting. We developed six different ELISAs using the six individual r-CFPs and one additional ELISA i.e. cocktail ELISA (c-ELISA) was prepared using cocktail of all 6 r-CFPs. The performance of all seven ELISAs were further evaluated against whole cell protoplasmic based indigenous ELISA (i-ELISA). c-ELISA showed almost similar sensitivity as shown by i-ELISA. However, individual r-CFP based ELISA could not reach up to the sensitivity of cocktail of six r-CFPs. None of the r-CFP showed any false positive (as compare to i-ELISA) thereby specificity was 100%. Results of ELISA tests based on cocktail of r-CFPs, ModD and 85C were quite similar to i-ELISA from goat sera whereas in cattle serum c-ELISA was comparable with i-ELISA. Our study showed a comparable specificity of c-ELISA for the diagnosis of JD and it may have applicability in region where disease is endemic. Future validation of c-ELISA against gold standard or confirmatory tests would give a better insight on its diagnostic potential over i-ELISA. Page(s): 219-229

Effect of Panax notoginsenoside Rg1 on bidirectional regulation of blood glucose level in mice

2021-04-01T00:00:00Z

Title: Effect of Panax notoginsenoside Rg1 on bidirectional regulation of blood glucose level in mice Authors: Guo, ShouBin; Chen, GuangHui; Song, Xingfa Abstract: Panax notoginseng saponin (PNS) is one of the key bioactive components of dry root and rhizome of Panax notoginseng (Burk.), a well known as Tianqi in Traditional Chinese Medicine (TCM). Although PNS has been shown to possess various pharmacological activities, such as being antithrombotic, neuroprotective, anti-inflammatory and hypolipidemic, etc., its effects on blood glucose levels have not been well documented but for some preliminary reports. It deserves a detailed in vivo investigation in animal model. Thus, to investigate the bi-directional regulation of ginsenoside Rgl (Rg1) on blood glucose in mice, Rg1 with high purity was prepared from panax notoginseng saponins by normal phase silica gel column chromatography and reverse phase C18 preparative chromatography. Normal 4-week-old mice were randomly divided into normal control group, normal control group with Rg1, glucose gavage control group, glucose gavage control group with Rg1, insulin treated control group and overnight fasting control group with or without Rg1 (n = 10). The mice in the control group were intragastrically administered with PBS solution, and the mice in the Rg1 groups were intragastrically administered with Rg1 once a day at the doses of 0.5, 1.0 and 1.5 mg/kg for consecutive 7 days. After the last drug, blood glucose (BG) levels were measured at 0.5 (30 min) and 1 h after administration using a simultaneous automatic biochemical analyzer to observe the effect of Rg1 on BG levels. Compared with the model group, Rg1 significantly decreased the BG levels of hyperglycemic mice induced by glucose gavage (P <0.05), significantly increased the BG level of overnight fasted mice (P <0.05), and had no significant effects on the normal group of mice. Panax notoginseng saponin Rgl has a significant bidirectional regulatory effect on glucose levels in mice. When the blood glucose of mice increases, intragastric administration of Rg1 can effectively reduce the blood glucose; conversely, when the blood glucose of mice is low, Rg1 can effectively increase the blood glucose value. Page(s): 230-235

Wound healing potency of Hemigraphis alternata (Burm.f) T. Anderson leaf extract (HALE) with molecular evidence

2021-04-01T00:00:00Z

Title: Wound healing potency of Hemigraphis alternata (Burm.f) T. Anderson leaf extract (HALE) with molecular evidence Authors: Sreekumar, Divyaa; Bhasker, Salini; Devi, Ponnuswamy Renuka; Mohankumar, C Abstract: Hemigraphis alternata (Burm.f) T. Anderson, commonly called as Red Ivy or Purple waffle plant and locally, Murikootti (Malayalam), is well known traditionally for its therapeutic effect in wound healing. Besides the analytical and biochemical evidence, more investigations at molecular level is inevitable for highlighting the healing effect of the herbal drug. In the present study, we have demonstrated the wound healing effect of H. alternata by in vitro cell line and animal model experiments using the Hemigraphis alternata leaf extract (HALE). The cell survival ability, cell proliferation and migration of cells in scratch wounds leading to wound closure were evident from the data by HALE treatment. Based on the wound closure observed in scratch wounds, the marker genes of wound healing PAI-1 and TGF-β1were analyzed by qPCR. The concentration and the expression fold were determined in 3T3-L1 and L6 cell lines treated with HALE. The concentration of marker gene of wound healing PAI-1 and TGF-β1showed significant increase in cell line cultures of 3T3-L1 and L6 cells treated with different concentrations of HALE (P <0.05). Similarly, the expression fold of both the genes was found higher in cells treated with HALE than the control. The molecular data showing the concentration and the expression of marker genes during wound healing in scratch wounds provide strong support for using HALE as a herbal medicine with the anti-inflammatory property and the antioxidant activity. Page(s): 236-245

Spatial distribution and sequential sampling of aphid and their natural enemies on wheat

2021-04-01T00:00:00Z

Title: Spatial distribution and sequential sampling of aphid and their natural enemies on wheat Authors: Alawi, Sayed Hazrat; Suroshe, Sachin S.; Chander, Subhash; Nebapure, Suresh M.; Poddar, Namita Abstract: Although many pests attack wheat, the damage due to wheat aphids has been on rise due to climate change phenomenon. Population of aphids increases during February to March and so the ladybird beetles to devour on aphids. With the aim of facilitating conservation of ladybird beetles, predator conditioned sampling plans were worked out. Wheat aphids, Rhopalosiphum padi L. and Sitobion avenae F. were observed infesting wheat. Major predatory coccinellid beetles, Cheilomenes sexmaculata (Fabricius) and Coccinella septempunctata (Linnaeus) were also found feeding on the wheat aphids. Spatial distribution of mixed population of the aphids and coccinellid beetles was analyzed using variance-mean ratio and regression models such as Taylor’s power law and Iwao’s mean crowding regression. The aphids and its predatory beetles were counted at different crop age. The mean population for wheat aphid was 7.88/plant and 0.72/ plant at 75 and 106 days after sowing (DAS), respectively. Variance-mean ratio indicated regular to aggregated distribution of aphids on the crop. Taylor’s power law aggregation parameter (b = 2.62) and density contagiousness co-efficient (β = 1.20) of Iwao’s mean crowding regression also revealed aggregated distribution of the wheat aphids. The mean population of coccinellid beetles varied from 0.72 to 1.43/plant during 68 to 100 DAS. Taylor’s power law aggregation parameter (b = 3.64) and density contagiousness co-efficient (β = 2.09) of Iwao’s mean crowding regression revealed aggregated distribution of the predators on the wheat crop. Sequential sampling plans were developed for wheat aphid management with and without predator’s effect through Taylor’s power law and Iwao’s mean crowding regression. Inclusion of predator effect in the regression models increased aphid population levels which necessitates need for management measures at higher population levels. Thus, the sequential sampling plans with predator effect are useful in avoiding unnecessary pesticide application on wheat crop for the aphid management. Page(s): 246-253