Detection and quantification of antibodies to Newcastle disease virus in ostrich and rhea sera using a liquid phase blocking enzyme-linked immunosorbent assay

A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method, the cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r = 0.875). The two tests showed good agreement ( = 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.

Cytotoxicity, genotoxicity and antimutagenicity of hexane extracts of Agaricus blazei determined in vitro by the comet assay and CHO/HGPRT gene mutation assay

Agaricus blazei Murrill ss. Heinem, known as the sun mushroom or himematsutake, is a basidiomycete native to Brazil, which is popular for its medicinal properties. The aim of this study was to test hexane extracts (one fraction and its four sub-fractions) of A. blazei for bioactivity in cultured mammalian cells (CHO-K1). The comet assay, the colony forming assay (CFA) and CHO/HGPRT gene mutation assay were used respectively to determine genotoxicity, cytotoxicity and antimutagenicity of these extracts at different concentrations. The cells were incubated in culture medium and treated for 3 h according to the standard protocol for each assay. The DNA damage-inducing agent ethylmethane sulfonate (EMS) was utilized as the positive control and also in combination with extracts to test for a protective effect. Statistical analysis of the data was performed using analysis of variance (ANOVA) and Tukey's test. A relationship between cytotoxicity and genotoxicity could be established and two extracts EH6B and EH6D showed a protective tendency, while the others did not, with the primary extract EH6 causing the most substantial damage to genetic material. These findings warrant more in-depth studies of the active principles of this mushroom. (c) 2005 Elsevier Ltd. All rights reserved.

Detection of cross infections by Leishmania spp. and Trypanosoma spp. in dogs using indirect immunoenzyme assay, indirect fluorescent antibody test and polymerase chain reaction

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

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)

Detection of T lymphocytes in intestine of broiler chicks treated with Lactobacillus spp. and challenged with Salmonella enterica serovar enteritidis

The expression of immune response in the form of leukocytic infiltrate by CD3+, CD4+, and CD8+ cells in the epithelium and in the intestinal lamina propria of chicks was studied in the present work by means of immunohistochemical reaction. The chicks were treated with Lactobacillus spp. or cecal microflora (CM) and experimentally challenged or not with Salmonella enterica serovar Enteritidis. The 320 birds utilized were divided into 4 groups containing 80 chicks each and submitted to treatments with Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus acidophilus, and CM. Each group was subdivided into 4 subgroups of 20 birds each and classified into a subgroup that did not receive treatment (negative control), subgroup treated, subgroup treated and challenged with Salmonella Enteritidis, and subgroup only challenged with Salmonella Enteritidis (positive control). The results obtained show that the treatment with L. reuteri, L. salivarius, L. acidophilus, or CM and challenged or not with Salmonella Enteritidis determine immune response in the form of leukocytic infiltrate by CD3+ and CD8+ lymphocytes followed by CD4+ in the epithelium and in the lamina propria of the duodenum, jejunum, and cecum of chicks up to 12 d of age. The quantity of CD3+ lymphocytes was significantly higher (P < 0.05) in the intestine of chicks treated with L. acidophilus or CM and challenged or not with Salmonella Enteritidis; however, the higher quantity of CD8+ lymphocytes was in the intestine of chicks treated with CM and challenged with Salmonella Enteritidis. The duodenum was the segment in which the immune response by T cells (CD3+, CD4+, and CD8+) was stimulated with the greatest intensity, followed by, respectively, the jejunum and cecum. The quantity of CD3+ lymphocytes present in the duodenum, jejunum, and cecum increases with the age of chicks, independent of the stimulus determined by treatments or challenge.

Evaluation of Salmonella-Lytic Properties of Bacteriophages Isolated from Commercial Broiler Houses

Because of recent interest in bacteriophage therapy in poultry, information regarding the interaction of bacteriophages and potential host bacteria in the environment should be collected. The present studies were initiated with a rather typical commercial broiler integrator within the south-central United States to examine environmental Salmonella levels in two broiler complexes, attempt to isolate Salmonella-lytic bacteriophages, and elucidate a possible reason for differing apparent Salmonella prevalence. Significantly ( P<0.05) less Salmonella was isolated from houses in complex 1 ( 15/44 [ 34%] Salmonella-positive drag swabs) as compared to houses in complex 2 ( 22/24 [ 92%]). A total of seven Salmonella-lytic bacteriophages were isolated from Salmonella-positive environments, and two bacteriophages were isolated from a single Salmonella-negative house. During the initial bacteriophage isolation, individual bacteriophages did not replicate in the Salmonella host isolated from the same environment, and lysis of additional Salmonella hosts relied on high numbers of bacteriophage to be present. This suggests that the presence of these bacteriophages in the environment of a commercial broiler house had little to no effect on the presence of Salmonella. This study highlights the need to find additional bacteriophage sources, more effective isolation methods, and more innovative approaches to using bacteriophages to treat enteric disease.

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.