Validation of an immunohistochemistry assay to detect turkey coronavirus: A rapid and simple screening tool for limited resource settings

The objective of the present study was to develop and apply the direct immunohistochemistry (D-IHC) assay to search for turkey coronavirus (TCoV) antigens in formalin-fixed embedded-paraffin tissues by the use of biotin-labeled polyclonal antibody. Twenty-eight-day-old embryonated turkey eggs (n = 50) were inoculated with TCoV-purified virus, and 3 d after inoculation, sections from ileum, ileum-cecal junction, and ceca were harvested, fixed in neutral formalin, and embedded in paraffin blocks and used as positive control. In addition, a total of 100 field samples from ileum, ileum-cecal junction, and ceca, collected from 30 to 45-d-old turkeys poults experiencing an outbreak of acute enteritis, were used to search for TCoV by the same D-IHC. All results were compared with those obtained by conventional RT-PCR and indirect fluorescent antibody assay (IFA) for all tested samples. Turkey coronavirus was detected in experimentally infected embryo tissues and also in field samples in 100% of ileum-cecal junction and ceca by the 3 detection procedures. With IFA as a reference assay, sensitivity and specificity of D-IHC were 98 and 58%, whereas sensitivity and specificity of reverse transcription-PCR were 96 and 66%, calculated from the total of tested samples from experimental infection. Each of the examined procedures was highly specific (D-IHC, 93%; RT-PCR, 90%), sensitive (D-IHC, 85%; RT-PCR, 86%), and agreement of both D-IHC and RT-PCR was 99 and 100%, respectively, compared with IFA results obtained from all the field samples. These findings demonstrated the utility of D-IHC for direct detection of TCoV from field samples and considering the sensitivity and specificity found here, can be used as an alternative technique.

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to pyrazinamide

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Comparative study of the efficacies of nine assay methods for the dextransucrase synthesis of dextran

A comparative study of nine assay methods for dextransucrase and related enzymes has been made. A relatively widespread method for the reaction of dextransucrase with sucrose is the measurement of the reducing value of D-fructose by alkaline 3,5-dinitrosalicylate (DNS) and thereby the amount of D-glucose incorporated into dextran. Another method is the reaction with C-14-sucrose with the addition of an aliquot to Whatman 3MM paper squares that are washed three times with methanol to remove C-14-D-fructose and unreacted C-14-sucrose, followed by counting of C-14-dextran on the paper by liquid scintillation counting (LSC). It is shown that both methods give erroneous results. The DNS reducing value method gives extremely high values due to over-oxidation of both D-fructose and dextran, and the C-14-paper square method gives significantly low values due to the removal of some of the C-14-dextran from the paper by methanol washes. In the present study, we have examined nine methods and find two that give values that are identical and are an accurate measurement of the dextransucrase reaction. They are (1) a C-14-sucrose/dextransucrase digest in which dextran is precipitated three times with three volumes of ethanol, dissolved in water, and added to paper and counted in a toluene cocktail by LSC: and (2) precipitation of dextran three times with three volumes of ethanol from a sucrose/dextransucrase digest, dried, and weighed. Four reducing value methods were examined to measure the amount of D-fructose. Three of the four (two DNS methods, one with both dextran and D-fructose and the other with only D-fructose, and the ferricyanide/arsenomolybdate method with is-fructose) gave extremely high values due to over-oxidation of D-fructose, D-glucose, leucrose, and dextran. (C) 2011 Elsevier Ltd. All rights reserved.

Mutagenic and genotoxic effects of the Atrazine herbicide in Oreochromis niloticus (Perciformes, Cichlidae) detected by the micronuclei test and the comet assay

Atrazine is the triazinic herbicide most found in the rural aquatic environments due to its extensive use and its stability in such places. The mutagenicity and the genotoxicity of different concentrations of the Atrazine herbicide were determinated by the micronucleus test and the comet assay, using Oreochromis niloticus as test-system. The tested concentrations of Atrazine herbicide were 6.25, 12.5 and 25 mu g/L, both for the micronuclei test and for the comet assay. The results showed a significant rate of micronuclei and nuclear abnormalities for all the tested concentrations of Atrazine herbicide. For the comet assay, we also observed results significantly different from the control in 6.25, 12.5 and 25 mu g/L concentrations. Due to these results, we could infer that such herbicide may be dangerous to the lives of those organisms exposed to it. (c) 2007 Elsevier B.V. All rights reserved.

Characterization of myeloid or lymphoid acute leukemia by a chemiluminescence assay: Comparison with immunocytochemistry using an antimyeloperoxidase antibody

A simple and sensitive chemiluminescence assay for the demonstration of the activity of intracellular myeloperoxidase (MPO) is described, which is useful for the distinction between myeloid and lymphoid commitment in blasts from acute leukemia patients. When the cut-off point was settled at 13 mV of chemiluminescence all cases of acute myeloid leukemia (AML) were distinguished from those of acute lymphoid leukemia. In addition, this technique was able to demonstrate MPO activity in AML poorly differentiated (FAB-M0) which usually does not stain for MPO in classical cytochemistry preparations and could be negative also by immunocytochemistry with anti-MPO monoclonal antibody. Therefore the method here described presented a higher sensitivity than the immunocytochemistry procedure with anti-MPO.

An enzyme-linked immunosorbent assay (elisa) for the detection of antibodies against babesia boris in cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Anesthesia of fish with benzocaine does not interfere with comet assay results

Fish blood erythrocytes are frequently used as sentinels in biomonitoring studies. Usually, fish blood is collected by painful cardiac or caudal vein punctures. Previous anesthesia could decrease animal suffering but it is not known at present whether anesthesia can cause confounding effects. Therefore, using the alkaline single cell gel (SCG)/comet assay with blood erythrocytes of the cichlid fish Nile tilapia, we tested for a possible modulation of induced DNA damage (methyl methanesulfonate; MMS) by the anesthetic benzocaine administered by bath exposure (80mg/l for ∼10min). Furthermore, benzocaine (80-600mg/l) was tested for its genotoxic potential on fish erythrocytes in vitro and for potential interactions with two known genotoxins (MMS and hydrogen peroxide). Our results did neither indicate a significant increase in the amount of DNA damage (even after a 48h follow-up), nor indicated interactions with MMS-induced DNA damage when fish were exposed to benzocaine in vivo. There was also no increase in DNA damage after in vitro exposure of fish erythrocytes to benzocaine. Clear concentration-related effects were observed for the two genotoxins in vitro, which were not significantly altered by the presence of benzocaine. These results suggest that anesthesia of fish does not confound comet assay results and the use of blood samples from anesthetized fish can be recommended with regard to animal welfare. © 2002 Elsevier Science B.V. All rights reserved.

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of Mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.

Investigation of the Genotoxic Potential of the Waters of a River Receiving Tannery Effluents by Means of the in vitro Comet Assay

The comet assay has been described as an efficient tool for the detection of changes in the DNA molecule of cells exposed to contaminating agents in vivo and in vitro. The possible environmental contamination due to the persistence of chromium residues from tannery effluents was determined in the waters of the Córrego dos Bagres stream, Municipal district of Franca/SP, by the comet assay on CHO-K1 cells. Water samples were collected during the four seasons of the year 2001 at three distinct stations along the river. The data suggest that the comet test showed good sensitivity for the environmental monitoring of these waters and indicated that this test can be efficient for the determination of the quality of waters contaminated with effluents containing heavy metal residues such as chromium.

Immobilization of lipases and assay in continuous fixed bed reactor

Lipases are versatile enzymes regarding the range of reactions they catalyse and substrates on which they act. They are as well important as catalyst in organic synthesis. Their immobilization on appropriate supports confer them greater stability besides the possibility of operating in continuous reactors. In order to explore these abilities, the reactions involving hydrolysis of p-nitrophenyl acetate (PNPA) and transesterification of PNPA with n-butanol were chosen. Lipases from two different sources were assayed, namely: microbial (Candida rugosa, CRL, Sigma Type VII) and pancreatic (PPL, Sigma, Type 11). Two immobilization methods were also used, namely: 1) adsorption, using as support the following silica derivatives (150-300μm e 450μ): phenyl, epoxy, amino and without derivation, and 2) covalent binding, using glutaraldehyde as binding agent and silica amino as support. This later method led to better results. Hydrolytic activity was 6.1 U/gsupport for CRL and 0.97U/gsupport for PPL, and of transesterification, 2,8U/gsupport for CRL and 1,9U/gsupport for PPL. Stability of the immobilized enzyme as a function of temperature was evaluated for CRL at 40°C and 50°C and for PPL at 32°C and 40°C. The assays were initially carried out batchwise, both for soluble and immobilized enzymes, aiming to the obtention of parameters for the continues reactor. Lipases immobilized by covalent binding were used in the assays of operacional stability in continuos reactors. For PPL in aqueous medium, at 32°C, and CRL in organic medium at 40°C, both operating continuously, no significant loss of activity was detected along the analysis period of 17 days. In the case of CRL in aqueous medium at 40°C there was a loss of activity around 40% after 18 days. For PPL in organic medium at 40°C the loss was 33% after 20 days. Compairing both sources with each other, very different results were obtained. Higher activitiy was found for CRL, both for hydrolysis and for transesterification reactions, with higher stability in organic medium. PPL showed lower activity as well as higher stability in aqueous medium. The immobilization method by covalent binding showed to be the most appropriate. Immobilized lipases are therefore relatively stable both in aqueous and organic medium.