Comparison of performance indicators of MGIT primary culture to assess the impact of implementation of ISO 15189:2012 standards.

Performance indicators are key component and plays a major role for monitoring and continuous quality improvement of the test results. The NABL certificate of accreditation is issued in accordance with the standard ISO 15189:2012 requirements. As part of the accreditation process, the laboratory has acquired knowledge and implemented the quality system procedures. Present study analyzed the impact of the accreditation process on the "performance indicators" of MGIT primary culture and found that performance indicators have been improved significantly after implementation of NABL for almost all indicators which clearly indicate the importance of accreditation and implementation of quality procedures for reliability of valid test results.

10-[2-(Dimethyl-amino)eth-yl]-9-(4-methoxy-phen-yl)-3,3,6,6-tetra-methyl-3,4,6,7,9,10-hexa-hydro-acridine-1,8(2H,5H)-dione.

In the title compound, C(28)H(38)N(2)O(3), the central ring of the acridinedione system adopts a boat conformation, while one of the outer rings adopts a half-chair conformation and the conformation of the other outer ring is between a sofa and a half-chair. The acridinedione system is buckled, with an angle of 22.01 (3)°. The crystal packing comprises layers of mol-ecules laid parallel to the ac plane, being reinforced by an intermolecular C-H⋯O interaction.

Detection of antibodies to egg drop syndrome virus in chicken serum using a field-based immunofiltration (flow-through) test.

A simple, user-friendly, and rapid method to detect the presence of antibodies to egg drop syndrome 76 (EDS) virus in chicken sera based on an immunofiltration (flow-through) test was developed. Purified EDS virus antigen was coated onto nitrocellulose membranes housed in a plastic module with layers of absorbent filter pads underneath. Following addition of serum to be tested and washing, monoclonal antibodies or polyclonal serum to chicken immunoglobulin G (IgG) was used as a bridge antibody to mediate binding between EDS virus-specific IgG and protein A gold conjugate. The appearance of a pink dot indicated the presence of antibodies to EDS virus in the sample tested. The results could be obtained within 5-10 min. The developed immunofiltration test could detect antibodies in the sera of experimentally vaccinated chickens from 2 wk postvaccination. With field sera samples, this test was positive in samples having hemagglutination inhibition titers of 8 and above. This test has the potential to be used as a field-based kit to assess seroconversion in EDS-vaccinated flocks.

Engineering the microstructure and permeability of thin multilayer latex biocatalytic coatings containing E. coli.

The microstructure and permeability of rehydrated 20-100 microm thick partially coalesced (vinyl-actetate acrylic copolymer) SF091 latex coatings and a 118 microm thick model trilayer biocatalytic coating consisting of two sealant SF091 layers containing a middle layer of viable E. coli HB101 + latex were studied as delaminated films in a diffusion apparatus with KNO(3) as the diffussant. The permeability of the hydrated coatings is due to diffusive transport through the pore space between the partially coalesced SF091 latex particles. Coating microstructure was visualized by fast freeze cryogenic scanning electron microscopy (cryo-SEM). The effective diffusion coefficient of SF091 latex coatings (diffusive permeability/film thickness) was determined as the ratio of the effective diffusivity of KNO(3) to its diffusivity in water (D(eff)/D). Polymer particle coalescence was arrested by two methods to increase coating permeability. The first used glycerol with coating drying at 4 degrees C, near the glass transition temperature (T(g)). The second method used sucrose or trehalose as a filler to arrest coalescence; the filler was then dissolved away. D(eff)/D was measured as a function of film thickness; content of glycerol, sucrose, and trehalose; drying time; and rehydration time. D(eff)/D varied from 3 x 10(-4) for unmodified SF091 coatings to 6.8 x 10(-2) for coatings containing sucrose. D(eff)/D was reduced by the flattening of latex particles against the surface of the solid substrate, as well as by the presence of the colloid stabilizer hydroxyethylcellulose (HEC). When corrected for the flattened particle layer, D(eff)/D of HEC-free coatings was as high as 0.20, which agreed with the value predicted from analysis of cryo-SEM images of the coat surface. D(eff)/D decreased by one-half in approximately 5 days in rehydrated SF091 coatings, indicating that significant wet coalescence occurs after glycerol, sucrose, or trehalose are leached from the films. D(eff)/D of SF091 latex trilayer coatings containing viable E. coli HB101 cells decreased as cell loading was increased from 2.2 x 10(-2) for 64 g dry cell weight per liter of coat volume to 5 x 10(-3) for 151 g DCW/L of coat volume. The reduction in coating permeability with increasing cell loading is predicted by Maxwell's equation for D(eff)/D in periodic composites.

Comparison of virus isolation and polymerase chain reaction for diagnosis of peste des petits ruminants.

Oculonasal swabs and tissue samples collected from peste des petits ruminants (PPR) suspected sheep and goats were tested for presence of the virus of peste des petits ruminants (PPRV) or its RNA by reverse transcription-PCR (RT-PCR) and virus isolation (VI). Of 44 samples 31.8% and 40.9% were positive by VI and RT-PCR, respectively. The RT-PCR-positive samples were subjected to the nested PCR. Three of six samples positive by RT-PCR but negative by VI were negative by the nested PCR. The specificity and accuracy of the nested PCR were higher than those of the RT-PCR although the sensitivity of both tests were similar. Nucleotide sequencing of one nested PCR product revealed a 92% homology with the sequence available in the GenBank (Acc. No. Z37017).

Sequence analysis of infectious bursal disease virus isolates from India: phylogenetic relationships.

Prevalence of infectious bursal disease (IBD) among chickens in different parts of Tamil Nadu, India, has been studied by collection of bursal samples from suspected flocks and by performing reverse transcription-polymerase chain reaction (RT-PCR) for amplification of a specific product of 474 bp from the variable region of the VP2 gene. Among 53 bursal samples examined by RT-PCR, 40 showed a positive reaction. The amplified products were subjected to nucleotide sequencing and the obtained sequences were compared with those of IBD virus (IBDV) vaccine strain Georgia, the classical virulent strain 52/70 and the very virulent Japanese OKYM strain. Nucleotide homology data indicated that all the Tamil Nadu isolates showed homology ranging from 91 to 99.6% among themselves. When compared with the very virulent Japanese OKYM strain, four isolates grouped with that strain. Majority of the isolates clustered with the very the virulent OKYM strain as evident from phylogenetic analysis performed using the MEGA program. Comparison of the deduced amino acid sequences of IBDV isolates with those of the vaccine strain Georgia, the classical virulent strain 52/70 and the very virulent strain OKYM also revealed the presence of conserved serine-rich heptapeptide sequence in most of the isolates. Results of this study indicate that majority of the IBDV isolates are very virulent, which is evident from heavy mortality that has been reported in few flocks of poultry in spite of regular vaccination.

Production and characterisation of monoclonal antibodies to an Indian isolate of peste des petits ruminants virus.

Nine monoclonal antibodies (Mabs) were produced against an Indian isolate of peste des petits ruminants (PPR) virus. These Mabs were directed against the nucleo (N) protein and were of IgG1 isotype. The Mabs produced intranuclear or coarse granular cytoplasmic fluorescence in PPR virus infected Vero cells and did not exhibit any neutralising activity. The Mabs cross-reacted with five other local isolates of PPR virus in slot blot hybridisation, radio immunoprecipitation assay (RIPA) and fixed-cell enzyme linked immunosorbent assay (ELISA). Two of the nine Mabs cross-reacted mildly with the vaccine strain of rinderpest (RP) virus in slot blot hybridisation and fixed-cell ELISA but did not precipitate the N protein of RP virus in RIPA. The N protein specific Mabs will be highly useful in differential diagnosis of PPR from RP.

Molecular Characterization of Thermostable Newcastle disease virus Isolated from Pigeon.

The HN and L gene sequences of an Indian isolate of Newcastle disease virus was analyzed prior to and after exposure to 56 °C at tenth passage and fifteenth passage to study the variations at molecular level. In the HN gene sequence of progenitor and thermostable strain, substitution of K373I, F374L, M516R, D517V were considered to contribute to the increase in the stability of the protein. In the L gene of the thermostable strain, variations were observed at many positions and among these the substitutions at position P675H K677R, K893D, R1132K, had charged amino acids, and at L656A, F657V, F869L, T886I, M899I, G1131V, V1675L, had hydrophobic amino acids that could be related to increased stability of L protein at high temperatures. The changes in amino acid sequence in HN and L gene of the thermostable strain might render structural variations that might have contributed to the stability of the strain at higher temperature.

Calcium phosphate coupled Newcastle disease vaccine elicits humoral and cell mediated immune responses in chickens.

Calcium phosphate (CaP) particles were coupled with inactivated Newcastle disease virus (NDV) vaccine. The surface morphology of CaP particles coupled to NDV was found to be spherical, smooth and with a tendency to agglomerate. The mean (± SE) size of CaP particles was found 557.44 ± 18.62 nm. The mean percent encapsulation efficiency of CaP particles coupled to NDV assessed based on total protein content and haemagglutination (HA) activity in eluate was found to be 10.72 ± 0.89 and 12.50 ± 2.09, respectively. The humoral and cell mediated immune responses induced by CaP coupled NDV vaccine were assessed in comparison to a commercial live vaccine (RDV 'F'). CaP coupled NDV vaccine elicited prolonged haemagglutination inhibition (HI) and enzyme linked immunosorbent assay (ELISA) titres in the serum even at fourth and fifth week post-vaccination (PV), unlike RDV 'F' inoculated chickens whose titres declined to insignificant levels by this time. CaP coupled NDV vaccine could stimulate HI antibodies in tracheal washings and tears from second and first week PV, respectively. IgA ELISA antibodies were also seen in tracheal washings of these birds from third week PV and in tears from second week PV. CaP coupled NDV vaccine elicited cell mediated immune responses (CMI) from two to four weeks PV. The stimulation indices obtained after stimulation with specific antigen was not significantly different between CaP coupled antigen and live NDV virus except on first week PV. However, CaP coupled antigen did not cause suppression of lympo proliferation as indicated by statistically similar responses to mitogen, concanavalin A between the two groups. Overall, CaP coupled NDV vaccine elicited stronger and prolonged immune responses in comparison to the commercial live vaccine. No increase in the serum calcium and phosphorous levels were seen in CaP coupled NDV vaccine inoculated chickens.

The value of tissue imprint hybridization for rapid detection of infectious bursal disease virus from field outbreaks.

The use of a peroxidase labelled PCR generated probe followed by enhanced chemiluminescence hybridization assay detected infectious bursal disease virus directly from bursal imprints on a nylon membrane. Tissue imprint hybridization proved to be a simple, rapid and safe means of detecting IBD virus for screening large numbers of field samples. The PCR generated probe was highly specific for IBD virus and did not hybridize with cellular nucleic acids in control imprints. Tissue imprint hybridization was found to be a more sensitive method than conventional antigen detection assays.

Detection of egg drop syndrome virus antigen or genome by enzyme-linked immunosorbent assay or polymerase chain reaction.

Mouse monoclonal antibodies (mAbs) were produced against an Indian isolate of egg drop syndrome (EDS) virus and characterized. Four hybridoma clones were secreting mAbs that bound to a 100 kDa protein, presumably the hexon protein. These mAbs were found to cross-react with two other Indian isolates of EDS virus and to the reference UK 127 strain. Three of these mAbs were mapped to the same epitope compared with the other mAb (F8), which bound to a different epitope. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed using the F8 mAbs as capture antibody and polyclonal chicken serum against EDS virus as detection antibody. A polymerase chain reaction (PCR) was used to detect the EDS viral genome. Following experimental infection of oestrogen-treated chickens with EDS virus, cloacal swabs, oviduct, uterus and spleen were collected at different days post-infection and used in both AC-ELISA and PCR, directly and after a single passage in embryonated duck eggs. The sensitivity and specificity of antigen detection by AC-ELISA or PCR was 95% and 98%, respectively. For diagnosis of EDS viral infections, PCR is recommended due to its ease and the lack of requirement of prepared reagents such as mAbs or conjugates. We recommend that PCR be performed directly on boiled tissue homogenates. Any negative samples may be passaged in embryonated duck eggs and the allantoic fluids tested by PCR before a conclusive negative diagnosis is given.

A patch coating method for preparing biocatalytic films of Escherichia coli.

A method has been developed for immobilizing viable but nongrowing Escherichia coli in highly uniform patches. The patches consist of a thin layer of bacteria in acrylate vinyl acetate covered with a thin layer of the same polymer devoid of bacteria and sealed by the edges. This method permits study of immobilized cell physiology in biocatalytic films by the assay methods used for suspended cells. Large numbers of patches of immobilized E. coli can be generated on metal or polyester sheets. Those described here are 12.7 mm in diameter; in them the cell layer is 30 microm thick and contains more than 5 x 10(8) viable cells. The method allows the cell-plus-polymer layer and the polymer sealant to be varied in thickness from 5 to 60 microm and from 7 to 80 microm, respectively. No leakage of cells was detected from 87% of the patches during 15 days of rehydration. Culturability of the immobilized cells, released by shaking the cells out of the porous polymer layer, was 80% of pre coating culturability. E. coli beta-galactosidase activity and measurements of total RNA and DNA from immobilized and suspended cells indicated that cells immobilized in the thin polymer layer have higher specific beta-galactosidase activity and a slower total RNA degradation rate than suspended cells over 15 days.