Development and evaluation of a MAb based competitive-ELISA using helicase domain of NS3 protein for sero-diagnosis of bovine viral diarrhea in cattle and buffaloes

The aim of this study was to develop a competitive inhibition ELISA (CI-ELISA) for detection of antibodies to bovine viral diarrhea virus (BVDV) using the helicase domain of NS3 (non-structural) protein and monoclonal antibody (MAb) against it and to estimate its sensitivity and specificity using two commercial ELISA kits as independent references. The 45-kDa helicase domain of NS3 protein of BVDV was expressed in Escherichia coli and 18MAbs were developed against it. MAb-11G8 was selected for use in CI-ELISA on the basis of maximum inhibition (90%) obtained with BVDV type 1 infected calf serum. Based on the distribution of percent inhibition of known negative sera (n=166), a cut-off value was set at 40% inhibition. In testing 914 field serum samples of cattle (810) and buffaloes (104), the CI-ELISA showed a relative specificity of 95.75% and 97.38% and sensitivity of 96% and 94.43% with Ingenesa kit and Institut Pourquier kit, respectively. This study proved that the use of helicase domain of NS3 (45-kDa) is equally good as the whole NS3 protein (80-kDa) used in commercial kits for detection of BVDV antibodies in cattle and buffaloes.     

Quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass Dicentrarchus labrax

Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red‐spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture‐based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 μg mL^?1 (3 × 10⁵ TCID50 per mL), whereas for capture‐based ELISA, the sensitivity for the antigen in solution was 17 μg mL^?1 (35 × 10⁵ TCID50 per mL). The capture‐based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 10⁸ TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv‐specific IgM is required, or for use in samples where PCR is difficult to perform.     

Establishment and characterization of macrophage cell line from thymus of Catla catla (Hamilton, 1822)

A continuous cell line has been developed from thymus explants of Catla catla and the cells have been subcultured for 63 passages. The cells exhibited optimum growth at 30°C in L‐15 medium containing 15% foetal bovine serum. The cultured cells engulfed yeast cells and fluorescent latex beads. These cells produced reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharide and phorbol esters. The culture supernatant from the cultured cells had lysozyme activity and these cells demonstrated Fc receptors. Almost all the cells were positive for alpha‐naphthyl acetate esterase enzyme suggesting that the cells are of macrophage lineage and therefore, the cell line was designated as catla thymus macrophage (CTM) cell line. CTM cells formed aggregates around zoospores of Aphanomyces invadans, but were unable to inhibit the germination of spores. The karyotype analysis of CTM cells at 25th passage revealed a typical diploid model with 50 chromosomes per cell. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and cytochrome c oxidase subunit 1 confirmed that the CTM cell line originated from C. catla. This cell line should be useful for studying the role of macrophages in differentiation and maturation of thymocytes and can be a source of macrophage‐specific enzymes and cytokines.     

Design and implementation of agriculture research digital photo manager

Software tools for photo collection management are proliferating, but they are generally developed for personnel photo management and have limited searching and browsing functions. We implemented the Agriculture Research Digital Photo Manager (ARDPM) prototype to enable researchers, extension personnel and other users to efficiently manage, search and browse their research photo archives. Research Digital Photo Manager provides a set of visual Boolean query interfaces for multiple ways of organizing and viewing the same photo collection. It gives users powerful search capabilities. Using thumbnail display and an easy interface, ARDPM is designed to provide users powerful searching and browsing photo capabilities.     

Development of a PCR assay and marker-assisted transfer of leaf rust resistance gene <Emphasis Type="Italic">Lr58</Emphasis> into adapted winter wheats

Leaf rust resistance gene Lr58 derived from Aegilops triuncialis L. was transferred to the hard red winter wheat (HRWW) cultivars Jagger and Overley by standard backcrossing and marker-assisted selection (MAS). A co-dominant PCR-based sequence tagged site (STS) marker was developed based on the sequence information of the RFLP marker (XksuH16) diagnostically detecting the alien segment in T2BS·2BL-2^tL(0.95). STS marker Xncw-Lr58-1 was used to select backcross F₁ plants with rust resistance. The co-dominant marker polymorphism detected by primer pair NCW-Lr58-1 efficiently identified the homozygous BC₃F₂ plants with rust resistance gene Lr58. The STS marker Xncw-Lr58-1 showed consistent diagnostic polymorphism between the resistant source and the wheat cultivars selected by the US Wheat Coordinated Agricultural Project. The utility and compatibility of the STS marker in MAS programs involving robust genotyping platforms was demonstrated in both agarose-based and capillary-based platforms. Screening backcross derivatives carrying Lr58 with various rust races at seedling stage suggested the transferred rust resistance in adapted winter wheats is stable in both cultivar backgrounds. Lr58 in adapted winter wheat backgrounds could be used in combination with other resistance genes in wheat rust resistance breeding.     

Buffalo SCNT embryos exhibit abnormal gene expression of ERK/MAPK pathway and DNA methylation

Inhibition of ERK/MAPK pathway has been shown to decrease DNA methylation via down‐regulation of DNA methyltransferases (DNMTs) in several studies suggesting that this pathway plays an important role in regulation of DNA methylation. We examined the relative expression level of seven important genes related to ERK/MAPK pathway and DNMTs (DNMT1, DNMT3a and DNMT3b) by quantitative real‐time PCR in buffalo blastocysts produced by Hand‐made cloning and compared it with that in blastocyst‐stage embryos produced by in vitro fertilization (IVF). The expression level of six of seven genes related to ERK/MAPK pathway examined i.e., p21RAS, RAF1, AKT1, ERK2, PIK3R2 and c‐Myc was significantly higher (p < 0.05) in cloned than in IVF embryos. However, the expression level of FOS was lower (p < 0.005) in cloned than in IVF embryos. The relative expression level of DNMT3a and DNMT3b but not that of DNMT1 was significantly higher (p < 0.05) in cloned than in IVF embryos. These results indicate that the cloned embryos exhibit an abnormal expression of several important genes related to ERK/MAPK pathway and DNMTs. Although a direct link between ERK/MAPK pathway and DNMTs was not examined in the present study, it can be speculated that ERK/MAPK pathway may have a role in regulating the expression of DNMTs in embryos, as also observed in other tissues.     

Effect of plant growth regulators on nitrate uptake and its reduction in radish cotyledons

Pretreatment of radish (Raphanus sativus L.) seedlings with exogenous hormones in the absence of external nitrate, resulted in a system having enough hormone levels to mediate the responses or activate the metabolic pathways which are necessary for nitrate (NO₃) uptake and reduction. Effects of pretreatment of radish seedlings with KN, GA, and ABA on the induction of NO₃ transport and corresponding NR activity, upon exposure to NO₃ were investigated. A low NO₃, uptake rate was observed with hormones when compared to DW‐control, while its induction pattern exhibited a sort of biphasic kinetics. It was observed that each hormone treatment affected NO₃ uptake and reduction specifically at different ambient concentrations of NO₃. On the basis of this, the operation of different constitutive and inducible (CHATS, LATS, HATS, and IHATS) transport systems was resolved for different hormonal treatments. Further, the hypothesis that nitrate uptake has direct role on nitrate reduction was supported by all treatments, except KN where highest NR activity despite lowest NO₃ uptake, was recorded. The results with KN points towards enhanced NR mRNA and de novo synthesis of enzyme.     

An investigation of the effects of feeding beta-aminopropionitrile on the growth rate and intramuscular collagen content of broiler chicks

In two experiments, 4‐ and 9‐week‐old broiler chicks were fed on diets containing either 0.025% or 0.1% β‐aminopropionitrile for 1 week. In both experiments the higher level caused some retardation of growth. There was, however, no differential effect on the size of the pectoralis muscle or on its collagen content. Enzyme tests showed little evidence of gross liver or bone damage but intramuscular collagen became more susceptible to solubilisation.

It is probable that feeding the lathyrogen in low concentration will make intramuscular collagen more amenable to study.