Detection of aldrin and dieldrin in egyptian milk samples using a competitive ELISA

A number of milk samples collected in Egypt from different animal species and at different locations were analyzed for the presence of the organochloride pesticides aldrin and dieldrin. A simple competitive enzyme‐linked immunosorbent assay (ELISA) was used for the detection and quantification of aldrin and dieldrin in milk samples from different species: buffalo, cow, goat, sheep and donkey. Pesticides were detected in 62.5% (10/16) of the buffalo milk samples, 73.33% (11/15) of the cows’ milk samples, 25% (3/12) of the goats’ milk samples, 71.42% (5/7) of the sheeps’ milk samples and 66–66% (2/3) of the donkeys’ milk samples.     

Indirect competitive ELISA for the quantitative and qualitative analysis of aldrin/dieldrin in egyptian milk samples from different farm animal species

The development of a simple, indirect, competitive ELISA is described for the detection and quantification of aldrin/dieldrin in Egyptian milk samples obtained from farm animals of different species used in dairy production in Egypt (buffalo, cow, goat and sheep) and from donkey milk. Milk samples (53) were collected from 18 stations in 16 different governorates in Egypt. The detection limit for aldrin/dieldrin in milk was 10 ppb—5 ppm without enhancement. Aldrin/dieldrin was detected in the following number of milk samples: buffalo, 62.5% (10/16); cow, 73.33% (11/15); goat, 66.66% (8/12); sheep, 71.428% (5/7); and donkey, 66.66% (2/3). The values reflect the lipophilic nature of these pesticides. The assay is rapid, sensitive, simple to perform and excellent for screening.     

Application of an aldrin and dieldrin ELISA to the detection of pesticides in eggs

This paper details the results obtained when a number of egg samples, collected in Egypt from different races of chicken were analyzed for the presence of the organochlorine insecticides aldrin and dieldrin. A simple ELISA was used for the detection and quantification of aldrin and dieldrin. The test was modified for application in this high protein system. Pesticide was detected in 83–352% (14/17) of the samples at levels ranging from 0.006 to 0.7 ppm (0.006 to 0.7 μg ml^‐1). People eating eggs containing these amounts of pesticides which are above the World Health Organisation average daily intake levels would be at increased risk.     

Development of an indirect competitive Elisa for Aldrin/Dieldrin in human milk samples collected in Egypt

Human milk samples (80) collected from 10 different cities in Egypt were tested for aldrin/dieldrin using an indirect enzyme‐linked immunosorbent assay (ELISA). Pesticides were detected in 73 of the 80 samples (91.25%) at levels ranging from 0.005 to 28 μg ml^‐1. These results present evidence for the persistence (or continued use) of these pesticides in Egyptian agriculture and their transmission through the food chain. More significantly the levels of pesticide in maternal milk (0.006–28 ppm) represent an unacceptably high level for infant intake; the maximum average daily intake is 0.0001 mg kg^‐1 body weight (WHO, 1972).     

Detection of aldrin/dieldrin in milk by competitive enzyme‐linked immunosorbent assay (ELISA)

Polyclonal antibodies raised against 6,7‐dihydro‐6‐carboxyaldrin can be used to detect aldrin and dieldrin. These analytes are preferentially fat soluble. An immunoassay is described which provides a method for detecting these pesticides in milk, a fat‐rich matrix. The enzyme‐linked immunosorbent assay (ELISA) can detect aldrin/dieldrin in milk in the range 1 ng ml^‐1‐5 μg ml^‐1 simply and reliably. The detection range differs in skimmed and semi‐skimmed milk and in cream, reflecting the differences in fat content between these samples.     

First identification of Naegleria species and Vahlkampfia ciguana in Nile water,Cairo, Egypt: Seasonal morphology and phylogenetic analysis

Background/PurposeIn Egypt, there is a scarcity of data concerning Naegleria (N.) family, with a shortage of phylogenetic studies. This study's aim was molecular detection, sequencing and phylogenetic analysis of morphologically identified Nagleria and to determine natural seasonal distribution of Nagleria species in water sources of Greater Cairo, Egypt.MethodsA total of 120 water samples were collected during each season over a year. Every water sample was filtrated and cultured on non-nutrient agar (NNA). Morphologically positive Nagleria-like isolates were subjected to Nagleria genus and species-specific PCR targeting rDNA gene, PCR products were sequenced and obtained sequences were phylogenetic analyzed. Results: Nile River water was the only source found to contained Naegleria. For the first time in Egypt, Vahlkampfia ciguana and the Naegleria species N.australiensis, N.philippinensis and N.neojejuensis were identified from the Nile water. The pathogenic Naegleria fowleri, previously reported in Egypt, was however not detected in this study.ConclusionInterestingly, there were no seasonal variations in prevalence of Naegleria spp.; yet, there was seasonal diversity in the water samples of the same site. These newly discovered Vahlkampfiidae in Egyptian aquatic environments indicate the need for further phylogenetic investigations using bigger sample sizes in order to determine their potential risk for human health.     

Detection of dieldrin in milk by (ELISA)

Polyclonal antibodies against an aldrin/dieldrin immunogen have been raised in rabbits and used as the basis of an enzyme‐linked immunosorbent assay (ELISA). This assay can detect dieldrin in milk in the range 5 μg ml ^‐1 to Ing ml ^‐1 reliably. This range differs in skimmed and semi‐skimmed milk, and in cream, reflecting the differences in fat content between these samples.     

A survey of residues of oganochlorine pesticides in hen eggs in Uganda

One hundred hen eggs were analysed by gas chromatography for the presence of residues of organochlorine pesticides. Sixty one eggs were collected from free-range hens in Tororo county in Tororo district and 39 eggs from enclosed hens (commercial layers) in the outskirts of Kampala city in Uganda. Residues of 9 organochroline compounds were detected in the hen eggs in the following frequencies: p,p'-DDE (100%), p,p'-DDT (85%), lindane (13%), dieldrin (11%), o,p'-DDT (10%), p,p'-DDD (8%), HCB (7%), &-HCH (4%), and o,p'-DDD (1%). All the mean residue levels were below the respective EEC MRL and FAO/WHO ERL. Only 3 (3%) samples contained total DDT levels above the EEC MRL and FAO ERL of 0.1mg /kg fresh weight. The residue levels of other organochlroine compounds in individual samples were below both the EEC MRL and the FAO/WHO ERL. It was concluded that contamination of hen eggs with residues of organochlorine pesticides from areas In Uganda that were considered In this study presents a low health risk to the consumers, and that organochlorine residue levels in hen eggs in Uganda were relatively lower than those reported from other African countries.     

Correlation between maternal milk and infant serum levels of chlorinated pesticides (CP) and the impact of elevated CP on bleeding tendency and immune status in some infants in Egypt

Chlorinated pesticides (CP) are environmentally persistent pollutants that (prenatally through the placenta and post-natally via breastfeeding) are transferred from mother to child. Considering the significant bleeding tendency noted in infants of CP-intoxicated mothers in Egypt, this study aimed to investigate any correlation between levels of these xenobiotics in mothers' milk and bleeding tendencies of their infants, as well as a possible role of any related immunosuppression in this phenomenon. This study examined 180 newborns presenting with altered bleeding tendencies and their mothers, and 180 normal newborns and their mothers (serving as a controls), selected from the Breastfeeding Unit, Center for Social and Preventive Medicine at the Cairo University Pediatric Hospital. Chlorinated pesticides (e.g., hexachlorocyclohexane, DDT, hepta-chloroepoxide, α- and β-endosulfan, aldrin, endrin, dieldrin) levels and their derivatives were measured in mothers' milk as well as in serum of neonates using gas chromatography/high resolution mass spectrometry. To link bleeding tendency with lactational intoxication of neonates by CP, newborns' blood was assessed for: platelet count, bleeding and prothrombin time, liver enzymes, Vitamin K, TNFα, and IL-10. Breast milk CP levels were associated with a higher incidence of bleeding in infants. Interference with the coagulation cascade was supported by changes in prothrombin time (prolonged), platelet counts (decreased), liver enzymes (increased), and serum vitamin K concentrations (decreased). Moreover, the significant decrease in WBC count and lymphocytes added to depressed cytokine secretion, i.e., TNFα and IL-10, suggested an organochlorine-induced immunotoxicity in infants developmentally exposed to the agents. We conclude that maternal transfer of CP, via breastfeeding or across the placenta, was sufficient to achieve similar CP levels in the serum of their infants; this correlated with a manifesting of altered bleeding tendencies and perturbed cytokine biology in these infants.     

Endotoxin and bacterial contamination of dialysis center water and dialysate; a cross sectional survey

The bacterial and endotoxin levels of purified water and effluent dialysate were examined in a cross section of dialysis centers in the central United States. All samples were collected within a four-hour drive of the University of Louisville and were collected, processed and analyzed by our personnel, to eliminate variability in sample handling. A medium capable of higher bacteria recovery from aqueous environments than those ordinarily employed in clinical assays was used. Endotoxins were determined by a quantitative colorimetric assay. By the more sensitive bacterial assay 53% of the centers had bacterial counts above the AAMI standard of 200 colony-forming units per ml (CFU/ml) for water and 35% of the centers had bacterial counts above the 2000 CFU/ml standard for dialysate in at least one sampling period. The samples showed 35% and 19% of water and dialysate above the standards, respectively. While there are no standards for endotoxin concentrations in water used to prepare dialysate, 2% of the centers had endotoxin levels in their water above five endotoxin units per ml (5 EU/ml = 1 ng/ml in our assay kit), the limit set by the AAMI standards for reprocessor water. Both bacterial and endotoxin levels tended to be elevated in dialysate, with the highest levels of endotoxin in dialysates posing an obvious potential risk when high-flux dialyzers are used.     

Use of the Comet test and micronucleus assay on human white blood cells for in vitro assessment of genotoxicity induced by different drinking water disinfection protocols

Surface water disinfection can lead to the formation of mutagenic/carcinogenic by-products derived from reactions with naturally occurring inorganic compounds. We investigated the feasibility and potential usefulness of an integrated approach to genotoxicity analysis of drinking water. The approach employed the Comet and micronucleus (MN) assays to evaluate the DNA and chromosomal damage produced by water extracts in human blood cells. Surface water samples from Lago Trasimeno (Italy) were collected in different seasons (July 2000, October 2000, February 2001, and June 2001), and samples were disinfected with sodium hypochloride (NaClO), chlorine dioxide (ClO(2)), or peracetic acid (PAA). Extracts of untreated and treated water were incubated with primary human leukocytes. The Comet assay revealed both strong seasonal variations and differences between samples processed by the three disinfection protocols. The three disinfectants increased the genotoxicity of the water collected in July 2000 and October 2000, with PAA producing the greatest amount of DNA damage. Extracts of raw water collected in February 2001 produced so much DNA damage that the relative genotoxic potentials of the three disinfectants could not be evaluated. No increase in MN frequency was detected in any of the samples. The multi-endpoint MN assay indicated, however, that our study samples (especially the sample collected in the February 2001) were cytotoxic. We conclude that this integrated approach to genotoxicity assessment may be useful both for the quality control of raw drinking water and to help compare the potential health risks associated with alternative disinfection processes.     

The role of oxidative stress in the in vitro induction of micronuclei by pesticides in mouse lung fibroblasts

The involvement of the antioxidant enzymes catalase and glutathione peroxidase (both at 0.1 mg/ml) in defence against the genotoxicity of phosphamidon (80 microg/ml) and dieldrin (25 microM) was investigated in order to demonstrate that the two pesticides damage DNA through the generation of reactive oxygen species and therefore of oxidative stress. The pesticide genotoxicity was determined by the cytokinesis-block micronucleus test performed on primary mouse lung fibroblast cultures. Also, 3-aminotriazole (40 mM) and mercaptosuccinate (0.5 mM), inhibitors of catalase and glutathione peroxidase, respectively, were added to the cultures. Data indicate that catalase causes a decrease only in the damage induced by phosphamidon, while glutathione peroxidase protects against damage induced by both phosphamidon and dieldrin. Simultaneous treatment with antioxidant inhibitors and pesticides results in a decrease in micronucleus frequency and cell number, due to apoptotic death. Our results indicate that clastogenic DNA damage produced by the two pesticides is modulated by antioxidant enzymes and their inhibitors and thus could be due to oxidative stress induction.     

Evaluation of a molecular beacon real-time PCR assay for detection of Baylisascaris procyonis in different soil types and water samples

Baylisascaris procyonis is a helminth parasite commonly found in North American raccoons (Procyon lotor) that is a cause of clinical neural, ocular, and visceral larva migrans in humans when infective eggs are ingested. Rapid detection of B. procyonis eggs in contaminated soil and water would assist public health analysts in evaluating risks associated with public exposure to areas of known raccoon activity. In this study, a molecular beacon probe-based real-time polymerase chain reaction (PCR) assay was developed to enable rapid and specific detection of eggs of Baylisascaris spp. The molecular beacon assay targeted the cytochrome oxidase subunit 2 (cox-2) gene of B. procyonis. To determine method sensitivity, experiments testing various egg levels (250, 25, and five eggs) were performed by seeding into 0.5-g soil samples or 0.5-mL water samples. Different soil sample types were extracted using a commercial nucleic acid extraction kit. Specificity testing using previously characterized helminth tissue specimens indicated that the assay was specific to Baylisascaris spp. Little real-time PCR inhibition was observed for most of the soil and water samples. A seed level of 250 eggs was detected for all soil types, and two seed levels (25 and five eggs) were detected for surface water samples. These results demonstrate that the reported real-time PCR assay was effective for the sensitive detection of B. procyonis in a wide range of soil types, and should be a useful tool for investigations of soil or water potentially contaminated with eggs of this parasite.     

Immune responses in sprague-dawley rats exposed to dibutyltin dichloride in drinking water as adults

Organotins are used commercially as agricultural pesticides, antifouling agents, and stabilizers for polyvinyl chloride (PVC) pipe. Mono- and di-substituted methyl and butyltins, used in PVC pipe production, are of concern as they leach from supply pipes into drinking water and have been reported to cause multisystem toxicity, including immunotoxicity. As part of an ongoing study to evaluate immunotoxic effects of organotins, we assessed immune function in adult Sprague-Dawley (CD) rats after exposure to dibutyltin dichloride (DBTC). Individually-housed adult male and female CD rats were given drinking water containing 0, 10, or 25 mg DBTC/L (final concentration) in 0.5% Alkamuls for 28 days. Water bottles were changed and water consumption was monitored twice weekly and body weights (BW) were recorded weekly. Delayed-type hypersensitivity (DTH), primary and secondary antibody responses to sheep red blood cells, and natural killer (NK) cell activity were evaluated in separate groups of treated and control animals on day 29 of exposure. Water consumption was significantly decreased in both sexes at 25 mg DBTC/L. BW, immune organ weights, the DTH response, and NK cell activity did not vary by dose. Different results for antibody responses in male rats were obtained in two experimental replicates. In the first replicate, IgG was elevated at the highest dose whereas in the second replicate, IgM was suppressed. However, as these effects occurred at the high dose of 25 mg DBTC/L, which is a concentration a million times higher than levels of DBTC reported in drinking water, our data suggest that DBTC is unlikely to cause immunotoxicity at concentrations found in drinking water supplies.     

Sensitive Immunoassays for Methyl-Parathion and Parathion and Their Application to Residues in Foodstuffs

Immunoassays, capable of detecting 0.05 μg l^?1 and 0.5 μg l^?1, respectively, have been developed to detect the organophosphate pesticides, methyl-parathion and parathion. Using haptens based on derivatization of the phosphate ester of the methyl and ethyl forms in the target compounds, there was selectivity in detection of methyl-parathion and parathion, respectively, using the two assays. Antisera to methyl-parathion detected parathion with 25-20% cross-reaction, while the parathion antisera detected methyl-parathion with 30-40% cross-reaction in water. The only other commonly-used agrochemical that cross-reacted in the assays was fenitrothion, the 3-methyl derivative of methyl-parathion. The assays were applied to the analysis of residues of these pesticides in water and several food matrices representative of different classes following the extraction of residues using simple procedures. Methanol extracts of most fruits and vegetables tested (high-moisture, low fat foods), including green and blue grapes, cauliflower and cabbage, could be analysed directly in the methyl-parathion assay, as could rice and basmati rice (low-moisture, low fat foods). Methanol extracts of butter and milk (high-fat foods) provided interference, but this was overcome by either further dilution or using acetonitrile as the extractant. A coagulating reagent was used to remove matrix interference from strongly coloured foods (tea and spinach). The parathion assay was subject to greater matrix interference, so it was preferable to analyse parathion in samples on plates coated with methyl-parathion antibody. With these foods, and with water samples, near-quantitative recoveries of spiked methyl-parathion or parathion were usually obtained, while with high-fat foods (milk and butter) and strongly coloured foods recoveries were poorer.     

Detection and quantitation of infectious human adenoviruses and JC polyomaviruses in water by immunofluorescence assay

Human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) have been proposed as markers of fecal/urine contamination of human origin. An indirect immunofluorescence assay has been developed to quantify infectious human adenoviruses types 2 and 41 and JC polyomaviruses strain Mad-4 in water samples. The immunofluorescence assay was compared with other quantitative techniques used commonly such as plaque assay, tissue culture infectious dose-50 and quantitative PCR (qPCR). The immunofluorescence assays showed to be specific for the detection of infectious viruses, obtaining negative results when UV or heat-inactivated viruses were analyzed. The assays required less time and showed higher sensitivity for the detection of infectious viral particles than other cell culture techniques (1 log₁₀ more) evaluated. River water samples spiked previously with human adenoviruses and raw sewage samples were also analyzed using the proposed immunofluorescence assay as well as by qPCR. The results show quantitations with 2 log₁₀ reduction in the numbers of infectious viruses compared with the number of genome copies detected by qPCR. The immunofluorescence assay developed is fast, sensitive, specific, and a standardizable technique for the quantitation and detection of infectious viruses in water samples.     

Rapid determination of triazine contamination of fresh water supplies by immunoassay

A highly sensitive, competitive, enzyme‐linked immunosorbent assay (ELISA) was developed for the detection of five triazine herbicides in water samples. Low detection limits were achieved without the need to concentrate samples prior to analysis by ELISA. The limit of detection for ametryn, prometryn and prometon was 0–05 ng ml～’ while atrazine and propazine had a detection limit of 0.2 ng ml^‐1. The concentrations of analyte required to reduce zero standard absorbances by 50% (IC₅₀) far ametryn, prometryn, prometon, propazine and atrazine were 0.18, 0.18, 0.26, 0.48 and 0.37 ng ml^‐1 respectively. De‐ethylatrazine and simazine had respective ICsos of 4.0 and > 25.0 ng ml^‐1. Inter‐assay coefficients of variation were generally less than 10% and recoveries of triazines from fortified water samples were in the range 90–115% for all five triazine herbicides. The results demonstrate the ability of an enzyme immunoassay to detect five triazine herbicides at concentrations below or very close to the maximum concentration allowed under EC guidelines in drinking water (0.1 ng ml^‐1) for individual pesticides.     

Genotoxicity monitoring of small bodies of water using two species of tadpoles and the alkaline single cell gel (comet) assay

To monitor genotoxicity in small bodies of water (e.g., creeks, ponds, and drainage ditches) we examined tadpole erythrocytes of two species: Rana clamitans and Rana pipiens, using the alkaline single cell gel DNA electrophoresis (SCG) or “comet” assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a “comet with tail” formation. Fifty-six samples, a total of 606 tadpoles, from 18 sites in southern Ontario, collected between 1993 and 1995, were examined. Samples of R. clamitans tadpoles collected in 1994 and 1995, from regions with heavy agricultural activity, gave significantly higher (P < 0.001) DNA length to width ratios than samples of R. clamitans tadpoles collected from sites in the Bruce Peninsula and near the French River, which have little or no agriculture. Samples of R. pipiens tadpoles collected in 1994 from sites on the outskirts of Windsor, Ontario, sites which receive genotoxic inputs from nearby industries, gave significantly higher (P < 0.001) DNA ratios than samples from agricultural areas and the Bruce Peninsula. R. clamitans tadpoles showed significant annual variation in DNA damage which was greater in samples of tadpoles collected from agricultural areas than from the Bruce Peninsula. The higher levels of DNA damage in tadpoles collected from agricultural areas may be due to the pesticides used, and the increased variation in DNA damage in the same areas is likely due to the impact of crop rotation, including leaving fields fallow, the timing of rainfall, and/or the application of pesticides. R. clamitans tadpoles, especially those collected from agricultural areas, also showed significant seasonal variation in DNA damage. There was no significant (P > 0.05) seasonal or annual variation in the levels of DNA damage in R. pipiens tadpoles collected from the Tallgrass Prairie. This study indicates that both species are suitable for use in the alkaline SCG assay and as in situ sentinel organisms for environmental biomonitoring. Environ. Mol. Mutagen. 29:418–430, 1997. © 1997 Wiley-Liss, Inc.     

Rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method

Primers for herpes simplex virus type 1 (HSV 1)-specific loop-mediated isothermal amplification (LAMP) method amplified HSV-1 DNA, while HSV-2-specific primers amplified only HSV-2 DNA; no LAMP products were produced by reactions performed with other viral DNAs. The sensitivities of the HSV-1- and HSV-2-specific LAMP methods, determined by agarose gel electrophoresis, reached 500 and 1,000 copies/tube, respectively. The turbidity assay, however, determined the sensitivity of the HSV-1- and HSV-2-specific LAMP methods to be 1,000 and 10,000 copies/tube, respectively. After initial validation studies, 18 swab samples (in sterilized water) collected from patients with either gingivostomatitis or vesicular skin eruptions were examined. HSV-1 LAMP products were detected by agarose gel electrophoresis in the 10 samples that also demonstrated viral DNA detection by real-time PCR. Nine of these 10 samples exhibited HSV-1 LAMP products by turbidity assay. Furthermore, both the agarose gel electrophoresis and the turbidity assay directly detected HSV-1 LAMP products in 9 of the 10 swab samples collected in sterilized water. Next, we examined the reliability of HSV type-specific LAMP for the detection of viral DNA in clinical specimens (culture medium) collected from genital lesions. HSV-2 was isolated from all of the samples and visualized by either agarose gel electrophoresis or turbidity assay.