A new technique for the recovery of Toxocara eggs from soil.

A new flotation technique for the extraction of Toxocara eggs from soil has been devised. It has a fairly high recovery efficiency for Toxocara eggs, can be used for replicated quantitative experiments, and may also be used for recovery of other ascarid eggs from soil.     

Optimized extraction of polycyclic aromatic hydrocarbons from contaminated soil samples

A comparison of Soxhlet extraction and a new extraction technique, fluidized-bed extraction, has been conducted. The extraction of polycyclic aromatic hydrocarbons (PAHs) by this new technique has been optimized considering as experimental variables the variation of the number of extraction cycles and the holding time after reaching the heating temperature by means of a surface response design. The significance of the operational parameters of the fluidized-bed extraction onto the performance characteristics has been investigated. For the determination of the analytes, a cleanup of the extracts followed by gas chromatography with mass spectrometric detection was used. The accuracy of the method was established by extraction and analysis of a reference material, supplied from the European Commission's Joint Research Centre.     

Improved methods for recovering eggs of Toxocara canis from soil

The ingestion of soil in parks and public places containing eggs of Toxocara may constitute a significant health risk, particularly to children. To determine the most efficient method for extracting eggs from experimentally contaminated soil, two consecutive studies were undertaken. Four techniques, including washing, sieving, vacuum, and the one recommended by the World Health Organization, were evaluated. Recovery rates of over 85% were recorded with both washing and sieving methods. Using the washing technique, all combinations of the four pre-treatment solutions, distilled water, acetoacetic solution pH 5, 0.1 n sodium hydroxide and 1% Tween 20, and seven flotation fluids with different specific gravities (S.G.) ranging from 1.20 to 1.35 were assayed. The association of distilled water and saccharose solution with an S.G. of 1.27 showed the best results, with a recovery rate of 99.91%.     

Cryptococcus spp. DNA extraction from environmental samples

The genus Cryptococcus encompasses 38 species, but only 3 are associated with disease in humans and animals, Cryptococcus laurentii, Cryptococcus albidus and Cryptococcus neoformans. The last one is the most frequently reported. The disease is acquired by the inhalation of infectious propagules present in the environment. The habitat has been established using extraction techniques with buffer supplemented with antibiotics and plating in selective media. The aim of this work was to evaluate several DNA extraction techniques for Cryptocococus spp. from environmental samples. The control isolates were C. neoformans, C. albidus, C. laurentii and Paracoccidiodes brasiliensis. We also used vermiculita and soil samples contaminated with different yeast concentrations (10 to 10(6) cells/g) and samples naturally contaminated with C. neoformans. DNA was extracted with physical and chemical methods and with a commercial kit, and the DNA was purified with agarose blocks and silica columns. For the PCR amplification we used the CN4-CN5 primers, which are specific for C. neoformans. Only the commercial kit allowed DNA extraction and amplification from contaminated soil samples up to a concentration of 10 cells/g and from one sample naturally colonized. With this work we extracted and amplified DNA from Cryptococcus spp. from environmental samples with appropriate PCR specificity, it will be a tool to establish the ecological areas of C. neoformans in our country.     

Loop-mediated isothermal amplification assay for detection and discrimination of Toxocara canis and Toxocara cati eggs directly from sand samples

We developed a novel and simple method, using loop-mediated isothermal amplification (LAMP), for the detection and discrimination of Toxocara canis and Toxocara cati eggs. The new method employs 4 steps: (1) concentration of Toxocara eggs in a small amount of sand; (2) dissolution of the proteinaceous membrane of eggs and simultaneously separation of them from the sand using NaClO treatment; (3) extraction of DNA using NaOH treatment; and (4) detection of T. canis / T. cati DNA using a LAMP assay. All these steps are fast, easy to perform, and do not require expensive equipment or reagents. The novel method was tested both experimentally and in a field study. In the laboratory, we could reliably detect as few as 3 T. canis eggs in artificially contaminated sand, if the experiment was repeated twice. In the field trial, we were able to detect T. cati DNA from 4 natural sandpits having moderate to heavy contamination, although not in a single lightly contaminated sandpit. All of the examined sandpits were found to be contaminated with eggs of T. cati, but none appeared to contain T. canis. Our new method could extract DNA from T. canis and T. cati eggs directly from sand samples as well as detect and distinguish these 2 species in a few easy steps, with markedly reduced time and expense.     

Decontamination by anaerobic stabilisation of the environment contaminated with enteronematode eggs Toxocara canis and Ascaris suum

Investigations were carried out under operating conditions of Field Composting Factory in Brezno (Slovak Republic) to determine the effect of anaerobic stabilization of organic wastes from public areas on the survival of model helminth Toxocara canis and Ascaris suum eggs. Due to anaerobic conditions, low temperature, low C:N ratio and changes in physical and chemical properties of organic waste, less than 64% of A. suum eggs remained viable after 150 days of stabilisation. The anaerobic stabilisation had a greater effect on the viability of T. canis eggs than on A. suum eggs. The infectivity of T. canis eggs was confirmed by a follow-up experiment in laboratory mice. A small number of T. canis larvae were found in their brain and muscles on day 28 after infection. The results refer to the risks of dissemination, survival and potential spread of endoparasitic developmental stages in the environment through organic wastes subjected to low temperature stabilisation.     

Methods for microbial DNA extraction from soil for PCR amplification

Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.     

PCR method based on the ogdH gene for the detection of Salmonella spp. from chicken meat samples

In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.     

3560例白带显微镜镜检法和阴道炎五联法检测结果比较分析

目的 探讨阴道炎五联检法与显微镜镜检法在白带常规检测中的应用比较及阴道炎五联检卡在白带检测中的应用价值.方法 3560例妇科门诊患者的白带同时用阴道炎五联检法和显微镜镜检法进行检测.结果 阴道炎五联检法与显微镜镜检法在白带常规检测中清洁度判断、乳酸杆菌、滴虫以及细菌性阴道病方面差异均无统计学意义(P＞0.05).在真菌的检测方面差异有统计学意义(P＜0.05).结论 阴道炎五联检卡操作简便、灵敏度高、专业的软件分析、结果科学准确,是白带检测较为理想的方法.     

Prevalence of Toxocara spp. eggs in some public grounds and highway rest areas in Kansas.

Out of a total of 282 soil samples obtained from public areas, 58 samples contained Toxocara spp. eggs. This gave an overall prevalence of 20.6%. Highway rest areas were contaminated and childrens sand boxes had the highest percentage of recovery of all.     

Utilizing a polymerase chain reaction method for the detection of Toxocara canis and T. cati eggs in soil

A polymerase chain reaction (PCR) technique has been used for the differentiation of T. canis and T. cati eggs isolated from soil and previously identified from microscopical observations. The method, using specific primers for the identification of the two Toxocara species, was assessed in both the field and laboratory. Successful results were obtained when only a single or large numbers of eggs were recovered from 40 g soil samples. The method is sensitive, allows analysis of material independent of the stage of egg development and can be adapted for the recovery of other species of parasites from soil.     

Prevalence and viability of eggs of Toxocara spp. and Toxascaris leonina in public parks in eastern Spain

To demonstrate the prevalence of Toxocara spp. and Toxascaris leonina eggs in parks in Murcia city, eastern Spain, a total 644 soil samples were examined from nine parks. More than 67% of parks and 1.24% of soil samples were contaminated and the mean egg density per sample was eggs per 100 g of soil. Over 97% of eggs identified were viable. Only one sample was positive for Toxascaris leonina. The present findings suggest that shady conditions are important for the occurrence and viability of Toxocara spp. and Toxascaris leonina eggs in soil as there were significantly more positive samples in shaded and moist areas compared with open and dry habitats.     

Phosphate solubilization by Penicillium spp. isolated from soil samples of Indian Himalayan region

A total of 246 fungal isolates representing 36 genera and 72 species were isolated from the soil samples collected from Indian Himalayan region. Twenty-one species belonged to the genus Penicillium alone. All the Penicillium species were screened for phosphate solubilizing activity on Pikovskaya agar at 21 °C. Eight species of Penicillium, exhibiting formation of halos (zone of solubilization) around the fungal colonies in qualitative plate assays, were selected for quantitative estimations. In quantitative estimations that were conducted upto day 30 (at 3 days interval), seven species of Penicillium brought maximum solubilization after day 15, while P. oxalicum showed maximum solubilization after day 21 of incubation. The increase in solubilization coincided with decrease in pH of the broth. Acid phosphatase activity was 1.5–2.0 times higher in comparison to alkaline phosphatase. Many of these species showed wide range of tolerance for temperature, pH and salt concentration.     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

Bioavailability and extraction of heavy metals from contaminated soil by Atriplex halimus

Pot experiments were performed to evaluate the phytoremediation capacity of plants of Atriplex halimus grown in contaminated mine soils and to investigate the effects of organic amendments on the metal bioavailability and uptake of these metals by plants. Soil samples collected from abandoned mine sites north of Madrid (Spain) were mixed with 0, 30 and 60 Mg ha⁻¹ of two organic amendments, with different pH and nutrients content: pine-bark compost and horse- and sheep-manure compost. The increasing soil organic matter content and pH by the application of manure amendment reduced metal bioavailability in soil stabilising them. The proportion of Cu in the most bioavailable fractions (sum of the water-soluble, exchangeable, acid-soluble and Fe–Mn oxides fractions) decreased with the addition of 60 Mg ha⁻¹ of manure from 62% to 52% in one of the soils studied and from 50% to 30% in the other. This amendment also reduced Zn proportion in water-soluble and exchangeable fractions from 17% to 13% in one of the soils. Manure decreased metal concentrations in shoots of A. halimus, from 97 to 35 mg kg⁻¹ of Cu, from 211 to 98 mg kg⁻¹ of Zn and from 1.4 to 0.6 mg kg⁻¹ of Cd. In these treatments there was a higher plant growth due to the lower metal toxicity and the improvement of nutrients content in soil. This higher growth resulted in a higher total metal accumulation in plant biomass and therefore in a greater amount of metals removed from soil, so manure could be useful for phytoextraction purposes. This amendment increased metal accumulation in shoots from 37 to 138 mg pot⁻¹ of Cu, from 299 to 445 mg pot⁻¹ of Zn and from 1.8 to 3.7 mg pot⁻¹ of Cd. Pine bark amendment did not significantly alter metal availability and its uptake by plants. Plants of A. halimus managed to reduce total Zn concentration in one of the soils from 146 to 130 mg kg⁻¹, but its phytoextraction capacity was insufficient to remediate contaminated soils in the short-to-medium term. However, A. halimus could be, in combination with manure amendment, appropriate for the phytostabilization of metals in mine soils.     

Clastogenicity of chromium contaminated soil samples evaluated by Vicia root-micronucleus assay.

Chromium compounds have been known to be highly toxic in biological systems and to some individuals act as strong allergens. A chromium processing plant in Tianjin city has been abandoned for many years and the chromium residue has been dispersed into the nearby soil. This study was designed to detect the genotoxicity of contaminated soil samples collected at various distances of 100 to 1000 m from the source using the Vicia faba root micronucleus test. Water solutions extracted from the soil samples were used to treat the roots of the Vicia beans. Micronuclei frequencies observed from the root meristems were used to determine the degree of genotoxicity. Micronuclei frequencies of the contaminated soil samples show linear dose responses to chromium contents in the soil, which were inversely proportional to the distance from the source.     

Public health implications of soil contaminated with helminth eggs in the metropolis of Kaduna, Nigeria

Environmental and socio-cultural variables influencing the distribution of helminth eggs in 608 soil samples were studied in 14 playgrounds that differ in socio-economic status in Kaduna metropolis, Nigeria, using a modified sieving method and a sucrose flotation medium of specific gravity 1.27. Helminth eggs were found in 62% of the soil samples and the distribution was as follows: Toxocara spp. 50.4%, Taenia spp./Echinococcus spp. 36.9%, Dipylidium caninum 26.3%, Ancylostoma spp. 9.0%, Ascaris spp. 7.2%, Trichuris spp. 3.7% and Ascaridia spp. 1.9%. A higher prevalence (68.1%) was recorded during the dry harmattan period while in the rainy period the rate was 58.1%. Mean egg densities ranged from 1.11 +/- 0.32 to 3.92 +/- 2.47 in areas moderately rated. Samples from site 14, which was highly rated, were more contaminated (78.1%) than those collected from other sites, while the intensity of contamination (14.0%) was more in moderately rated site 4 than in the rest of the sites. There were significant associations between the prevalence of helminth eggs and rainy period of the study (odds ratio (OR) = 0.38; 95% confidence interval (CI) on OR: 0.20 0.05). This study shows that the period of study, the presence of dogs and vegetation influence the prevalence of helminth eggs in soil in Kaduna metropolis.     

Quantitative PCR method for detection of mycoplasma spp. DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii)

Mycoplasma agassizii and M. testudineum have been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Because microbiological culture methods have proven difficult to employ in wild desert tortoises, our goal was to develop a sensitive and specific qPCR method for detecting and quantifying mycoplasma DNA in nasal lavage fluid collected in the field. Primers for 16S ribosomal RNA gene sequences specific for M. agassizii and M. testudineum were designed, together with primers that recognize conserved sequences of both microorganisms. Standard curves generated with DNA extracted from known numbers of mycoplasma cells revealed a lower detection limit of approximately 5 fg. The qPCR method did not recognize normal flora DNA, and nasal lavage fluid contained no interfering substances. Nasal lavage samples collected from 20 captive desert tortoises housed at the Desert Tortoise Conservation Center (Clark County, Nevada, USA) revealed the presence of M. agassizii DNA in 100% of the tortoises. Concentrations ranged from a low of 6 pg ml^− 1 to a high of 72,962 pg ml^− 1. Only one of the tortoises was positive for M. testudineum. Interestingly, not all of the qPCR positive tortoises showed evidence of seroconversion, suggesting that they were colonized but not infected. This new quantitative method will provide a critical tool for managing threatened populations of the desert tortoise.