Evolution of subpatent parasitaemia in Trypanosoma cruzi chronically infected mice with the help of a cyclophosphamide amplification transfer assay

Avaliamos o potencial do ensaio clássico de subinoculação, modificado pelo tratamento com ciclofosfamida dos animais receptores, na detecção de parasitemias ocultas em camundongos com in-fecção crônica pelo Trypanosoma cruzi. O ensaio, além de simples, mostrou ter uma alta sensibilidade; assim, utilizando-se parasitas da fase aguda, o tratamento com ciclofosfamida revelou parasitemias em 53,8% dos animais infectados com um tripanosoma da cepa y, e em 20% dos animais infectados com um tripanosoma da cepa CL. O tratamento com ciclofosfamida aumentou a sensibilidade do ensaio de subinoculação nas infecções pela cepa CL, e resultou em igual sensibilidade quando utilizada a cepa Y. Nos camundongos de fase crônica, obtidos a partir de diversos esquemas de imunoprofilaxia (BCG, soro de camundongo imune) ou quimioterapia, o ensaio revelou parasitemias ocultas em 99% dos animais. Auxiliados pelo método da subinoculação-ciclofosfamida estudamos no espaço de um ano a evolução das parasitemias ocultas em um grupo de camundongos infectados que sobreviveram à fase aguda pelo tratamento com Benzonidazol. O ensaio revelou parasitemias ocultas em 100% dos animais. Entretanto, padrões contínuos e discontinuos de positividade puderam ser detectados.

Genotoxicity and mutagenicity of water contaminated with tannery effluents, as evaluated by the micronucleus test and comet assay using the fish Oreochromis niloticus and chromosome aberrations in onion root-tips

Cytotoxicity of metals is important because some metals are potential mutagens able to induce tumors in humans and experimental animals. Chromium can damage DNA in several ways, including DNA double strand breaks (DSBs) which generate chromosomal aberrations, micronucleus formation, sister chromatid exchange, formation of DNA adducts and alterations in DNA replication and transcription. In our study, water samples from three sites in the Córrego dos Bagres stream in the Franca municipality of the Brazilian state of São Paulo were subjected to the comet assay and micronucleus test using erythrocytes from the fish Oreochromis niloticus. Nuclear abnormalities of the erythrocytes included blebbed, notched and lobed nuclei, probably due to genotoxic chromium compounds. The greatest comet assay damage occurred with water from a chromium-containing tannery effluent discharge site, supporting the hypothesis that chromium residues can be genotoxic. The mutagenicity of the water samples was assessed using the onion root-tip cell assay, the most frequent chromosomal abnormalities observed being: c-metaphases, stick chromosome, chromosome breaks and losses, bridged anaphases, multipolar anaphases, and micronucleated and binucleated cells. Onion root-tip cell mutagenicity was highest for water samples containing the highest levels of chromium.

Atrazine interaction with tropical humic substances by Enzyme Linked Immunosorbent Assay

Enzyme-Linked Immunosorbent Assay (ELISA) has been evaluated by analyzing rich-humic water samples from tropical rivers. The samples were spiked with atrazine at ppb level Different pHs (4 to 9) and humic concentrations (2.5 to 40 mg L-1) were investigated. The assay performance showed a strong dependence on the pH values and amount of humic matter at low atrazine concentration. From all the conditions studied the low pH (pH 4) and high humic substances concentrations (40 mg L-1) showed the greatest influence. The IC50 value to control sample (no humic) diminished from 0.28 nmol L-1 to 0.64 nmol L-1 to humic acid solution. This effect is specially noted for the humic acid fractions, since fulvic acid fractions showed no significant change on the immunoassay results. Additionally, it has been demonstrated that at basic pH the matrix effect produced by the natural Brazilian water sample containing humic substances even at 40 mg L-1 disappears. Therefore, the ELISA method used to determine atrazine, can be employed to determine this pesticide in water samples containing humic substances without prior preparation.

THE EFFECT OF CARBOHYDRATE ADMINISTRATION ON EXPERIMENTAL-INFECTION WITH SALMONELLA SEROTYPES IN CHICKENS

Carbohydrates and cultures of faecal microflora were administered to newly hatched chicks to prevent infection with Salmonella typhimurium Salmonella enteritidis, Salmonella agona and Salmonella infantis. Birds were killed 72 hours after challenge and the number of viable Salmonella organisms in their caecal contents estimated. Carbohydrates did not promote efficient control of infection with the Salmonella serotypes tested whereas cultures of faecal microflora completely prevented infection with all serotypes.

STUDIES ON THE USE OF A FORMIC-ACID PROPIONIC-ACID MIXTURE (BIO-ADD(TM)) TO CONTROL EXPERIMENTAL SALMONELLA INFECTION IN BROILER-CHICKENS

Experiments were carried out on the antibacterial effects of a commercial formic acid-propionic acid mixture (Bio-add(TM)) against different Salmonella serptj pes. The preparation exerted a strong antibacterial effect on S. typhimurium strain F98 in artificially contaminated feed. After 28 days storage, the bactericidal effect was still considerable. When chickens were reared on feed that had been treated with Bio-add(TM) and artificially contaminated with different serotypes, S. enteritidis, S. typhimurium and S. agona were not isolated from the caecal contents, but S. infantis was. No organisms of this strain were isolated when a lower feed-contamination rate of bacteria was used.

Mutagenic compounds generated from the chlorination of disperse azo-dyes and their presence in drinking water

The water produced by the Cristais River Drinking Water Treatment Plant (CR-DWTP) repeatedly produced mutagenic responses that could not be explained by the presence of disinfection byproducts (DBPs) generated by the reaction of humic acids and chlorine. In order to determine the possible role of chlorinated dye products in this mutagenic activity, solutions of a black dye commercial product (BDCP) composed of C. I. Disperse Blue 373, C. I. Disperse Orange 37, C. I. Disperse Violet 93, and chemically reduced BDCP (R-BDCP) were chlorinated in a manner similar to that used by the CR-DWTP. The resulting solutions were extracted with XAD-4 along with one drinking water sample collected from the CR-DWTP. All extracts showed mutagenic activity in the Salmonella/microsome assay. Dye components of the BDCP as well as its reduced chlorinated (Cl-R-BDCP) derivative were detected in the drinking water sample by analysis with a high performance liquid chromatography/diode array detector (HPLC/DAD). The mutagenicity results of these products suggest that they are, at least in part, accounting for the mutagenic activity detected in the drinking water samples from the Cristais River. The data obtained in this study have environmental and health implications because the chlorination of the BDCP and the R-BDCP leads to the formation of mutagenic compounds (Cl-BDCP and Cl-R-BDCP), which are potentially important disinfection byproducts that can contaminate the drinking water as well as the environment.

Assay of Serum Glutamic-Oxaloacetic Transaminase Using Malate Dehydrogenase Preparation Obtained from Streptomyces aureofaciens

A modified spectrophotometric method for serum glutamic-oxaloacetic transaminase (SGOT) assay was developed. A crude cell-free extract from Streptomyces aureofaciens which showed a high level of malate dehydrogenase (MDH) activity (E.C. 1.1.1.37) was used as the enzymatic indicator. The lyophilized microbial preparation was used without previous purification and was quite stable under refrigeration for one year. Serum sample assays using both the method utilizing the crude cell extract and an enzymatic commercial kit showed good correlation.

An enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Toxocara vitulorum in water buffaloes

Toxocara vitulorum, a parasite of the small intestine of cattle and water buffaloes, is mainly acquired by calves via the colostrum/milk from infected cows. To understand the development of immune responses in calves, antibody levels to a soluble extract antigen (Ex) from T. vitulorum infective larvae were measured by an indirect ELISA with sera of 15 buffalo calves, which were sampled every 15 days for the first 180 days after birth and 9 buffalo cows during the perinatal period. From all serum samples examined during the first 180 days, antibody level was lowest and highest in calves at 1 day of age before and after suckling colostrum, respectively, suggesting that the origin of antibodies was the colostrum. Immediately after birth, antibody levels in suckled calves remained at high levels until day 15, began to decrease to lower levels between 15 and 30 days and remained relatively stable until 120 days. By comparing the immune responses of these animals with their parasitological status it was considered possible to determine if passively acquired or actively produced antibodies provided protection against the infection. High numbers of T. vitulorum eggs in the feces between 30 and 60 days indicated that passively acquired antibodies did not provide protection against the infection, at least during these first days, and the maximum fecal egg counts during 30-45 days were coincident with decreased antibody levels. Between 60 and 120 days, when serum antibodies were detected at reduced, but stable levels, adult nematodes were expelled from the intestines and no more T. vitulorum eggs were found, suggesting development of acquired resistance. However, the potential and functional protective role of the antibodies against T. vitulorum infection and the process of self-cure requires further investigation. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Ácidos orgânicos e microbiota cecal anaeróbia no controle da infecção experimental de frangos por Salmonella typhimurium e Salmonella enteritidis

The aim of this investigation was to study the effect of organic acids and/or anaerobic cecal microflora (ACM) on systemic and digestive infection of broilers by Salmonella typhimurium and S. enteritidis. ACM was used without previous bacterial identification. The treatment with ACM increased the resistance to Salmonella spp infection. Infection was more evident in caeca, followed by rectum and crop and did not interfere on body weight of broilers. Treated and control groups showed the same degree of infection at the end of the experiment. The use of ACM isolated or combined with acetic acid, reduced the colonization of the chick's digestive system by S. typhimurium and S. enteritidis. Acetic acid added to ACM did not potentiate the reduction of digestive system colonization. Except for the crop, the isolated use of acetic, propionic or formic acids did not reduce S. typhimurium and S. enteritidis, in caeca and rectum. The use of organic acids and ACM had little effect on reduction of caecum pH. The treatment with ACM reduced the quantity of S. enteritidis in the faeces. The reduction of caecum pH did not reduce the quantity of S. enteritidis in faeces. S. enteritidis was much more invasive than S. typhimurium and use of organic acids and ACM had little effect on reduction of systemic infection.

Liquid-phase blocking sandwich enzyme-linked immunosorbent assay for detection of antibodies against foot-and-mouth disease virus in water buffalo sera

Objective-To develop and apply the liquid-phase blocking sandwich ELISA (BLOCKING-ELISA) for the quantification of antibodies against foot-and-mouth disease virus (FMDV) strains O-1 Campos, A(24) Cruzeiro, and C-3 Indaial.Design-Antibody quantification.Sample Population-158 water buffalo from various premises of São Paulo Stale-Brazil. The sera were collected either from systemically vaccinated or nonvaccinated animals.Procedure-The basic reagents of BLOCKING-ELISA (capture and detector antibodies, virus antigens, and conjugate) were prepared and the reaction was optimized and standardized to quantify water buffalo antibodies against FMDV. An alternative procedure based on mathematical interpolation was adopted to estimate more precisely the antibody 50% competition liters in the BLOCKING-ELISA. These titers were compared with the virus-neutralization test (VNT) titers to determine the correlation between these techniques. The percentages of agreement, cutoff points, and reproducibility also were determined.Results-The antibody liters obtained in the BLOCKING-ELISA had high positive correlation coefficients with VNT, reaching values of 0.90 for O-1 Campos and C-3 Indaial, and 0.82 for the A(24) Cruzeiro (P < 0.0005). The cutoff points obtained by use of the copositivity and conegativity curves allowed determination of high levels of agreement between BLOCKLNG-ELISA and VNT antibody titers against the 3 FMDV strains analyzed.Conclusions-The results characterized by high cor relation coefficients, levels of agreement, and reproducibility indicate that the BLOCKING-ELISA may replace the conventional VNT for detection and quantification of antibodies from water buffalo sera to FMDV.

DETECTION OF SALMONELLA-TYPHIMURIUM IN A BROILER CHICKEN FLOCK

An outbreak of Salmonella typhimurium in a commercial broiler chicken flock is reported. The signs of the disease started on the 5th day-old. The symptoms, the gross alterations and the damage to the birds and to the farm are discussed.

Use of comet assay to assess DNA damage in patients infected by Helicobacter pylori - Comparisons between visual and image analyses

Studies of DNA damage in gastric epithelial cells of Helicobacter pylori (H. pylori)-infected patients are conflicting, possibly due to different methods used for scoring DNA damage by Comet assay. Therefore, we compared the sensitivity of visual microscopic analysis (arbitrary units-scores and comets%) and image analysis system (tail moment), in the gastric epithelial cells from the antrum and corpus of 122 H. pylori-infected and 32 non-infected patients. The feasibility of cryopreserved peripheral blood lymphocytes and whole-blood cells for DNA damage biomonitoring was also investigated. In the antrum, the levels of DNA damage were significantly higher in H. pylori-infected patients with gastritis than in non-infected patients with normal mucosa, when evaluated by image analysis system, arbitrary units and comets%. In the corpus, the comets% was not sufficiently sensitive to detect the difference between H. pylori-infected patients with gastritis and non-infected patients with normal mucosa. The image analysis system was sensitive enough to detect differences between non-infected patients and H. pylori-infected patients with mild gastritis and between infected patients with moderate and severe gastritis, in both antrum, and corpus, while arbitrary units and comets% were unable to detect these differences. In cryopreserved peripheral blood lymphocytes, the levels of DNA damage (tail moment) were significantly higher in H. pylori-infected patients with moderate and severe gastritis than in non-infected patients. Overall, our results indicate that the image analysis system is more sensitive and adequate to measure the levels of DNA damage in gastric epithelial cells than the other methods assayed. (c) 2005 Elsevier B.V. All rights reserved.

Chemical characterization of a dye processing plant effluent - Identification of the mutagenic components

This work shows the chemical characterization of a dye processing plant effluent that was contributing to the mutagenicity previously detected in the Cristais river, São Paulo, Brazil, that had an impact on the quality of the related drinking water. The mutagenic dyes Disperse Blue 373, Disperse Orange 37 and Disperse Violet 93, components of a Black Dye Commercial Product (BDCP) frequently used by the facility, were detected by thin layer chromatography (TLC). The blue and orange dyes were quantified by high performance liquid chromatography (HPLC/DAD) in a raw and treated effluent samples and their contribution to the mutagenicity was calculated based on the potency of each dye for the Salmonella YG1041. In the presence of S9 the Disperse Blue 373 accounted for 2.3% of the mutagenic activity of the raw and 71.5% of the treated effluent. In the absence of S9 the Disperse Blue 373 accounted for 1.3% of the mutagenic activity of the raw and 1.5% of the treated effluent. For the Disperse Orange 37, in the presence of S9, it contributed for 0.5% of the mutagenicity of the raw and 6% of the treated effluent. In the absence of S9; 11.5% and 4.4% of the raw and treated effluent mutagenicity, respectively. The contribution of the Disperse Violet 93 was not evaluated because this compound could not be quantified by HPLC/DAD. Mutagenic and/or carcinogenic aromatic amines were also preliminary detected using gas chromatograph/mass spectrometry in both raw and treated and are probably accounting for part of the observed mutagenicity. The effluent treatment applied by the industry does not seem to remove completely the multagenic compounds. The Salmomella/microsome assay coupled with TLC analysis seems to be an important tool to monitor the efficiency of azo dye processing plant effluent treatments. (c) 2006 Elsevier B.V. All rights reserved.