A real-time PCR method for quantifying viable ascaris eggs using the first internally transcribed spacer region of ribosomal DNA

Worldwide, 1.4 billion people are infected with the intestinal worm Ascaris lumbricoides. As a result, Ascaris eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable Ascaris eggs is time- and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable Ascaris eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to Ascaris egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications.     

Inactivation of Single-Celled Ascaris suum Eggs by Low-Pressure UV Radiation

Intact and decorticated single-celled Ascaris suum eggs were exposed to UV radiation from low-pressure, germicidal lamps at fluences (doses) ranging from 0 to 8,000 J/m² for intact eggs and from 0 to 500 J/m² for decorticated eggs. With a UV fluence of 500 J/m², 0.44- ± 0.20-log inactivation (mean ± 95% confidence interval) (63.7%) of intact eggs was observed, while a fluence of 4,000 J/m² resulted in 2.23- ± 0.49-log inactivation (99.4%). (The maximum quantifiable inactivation was 2.5 log units.) Thus, according to the methods used here, Ascaris eggs are the most UV-resistant water-related pathogen identified to date. For the range of fluences recommended for disinfecting drinking water and wastewater (200 to 2,000 J/m²), from 0- to 1.5-log inactivation can be expected, although at typical fluences (less than 1,000 J/m²), the inactivation may be less than 1 log. When the eggs were decorticated (the outer egg shell layers were removed with sodium hypochlorite, leaving only the lipoprotein ascaroside layer) before exposure to UV, 1.80- ± 0.32-log reduction (98.4%) was achieved with a fluence of 500 J/m², suggesting that the outer eggshell layers protected A. suum eggs from inactivation by UV radiation. This protection may have been due to UV absorption by proteins in the outer layers of the 3- to 4-μm-thick eggshell. Stirring alone (without UV exposure) also inactivated some of the Ascaris eggs (~20% after 75 min), which complicated determination of the inactivation caused by UV radiation alone.     

A Quantitative Assessment Method for Ascaris Eggs on Hands

The importance of hands in the transmission of soil transmitted helminths, especially Ascaris and Trichuris infections, is under-researched. This is partly because of the absence of a reliable method to quantify the number of eggs on hands. Therefore, the aim of this study was to develop a method to assess the number of Ascaris eggs on hands and determine the egg recovery rate of the method. Under laboratory conditions, hands were seeded with a known number of Ascaris eggs, air dried and washed in a plastic bag retaining the washing water, in order to determine recovery rates of eggs for four different detergents (cationic [benzethonium chloride 0.1% and cetylpyridinium chloride CPC 0.1%], anionic [7X 1% - quadrafos, glycol ether, and dioctyl sulfoccinate sodium salt] and non-ionic [Tween80 0.1% -polyethylene glycol sorbitan monooleate]) and two egg detection methods (McMaster technique and FLOTAC). A modified concentration McMaster technique showed the highest egg recovery rate from bags. Two of the four diluted detergents (benzethonium chloride 0.1% and 7X 1%) also showed a higher egg recovery rate and were then compared with de-ionized water for recovery of helminth eggs from hands. The highest recovery rate (95.6%) was achieved with a hand rinse performed with 7X 1%. Washing hands with de-ionized water resulted in an egg recovery rate of 82.7%. This washing method performed with a low concentration of detergent offers potential for quantitative investigation of contamination of hands with Ascaris eggs and of their role in human infection. Follow-up studies are needed that validate the hand washing method under field conditions, e.g. including people of different age, lower levels of contamination and various levels of hand cleanliness.     

Inactivation of Viable Ascaris Eggs by Reagents during Enumeration

Various reagents commonly used to enumerate viable helminth eggs from wastewater and sludge were evaluated for their potential to inactivate Ascaris eggs under typical laboratory conditions. Two methods were used to enumerate indigenous Ascaris eggs from sludge samples. All steps in the methods were the same except that in method I a phase extraction step with acid-alcohol (35% ethanol in 0.1 N H₂SO₄) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-μm-mesh stainless steel sieve that retained the eggs. The concentration of eggs and their viability were lower in the samples processed by method I than in the samples processed by method II by an average of 48 and 70%, respectively. A second set of experiments was performed using pure solutions of Ascaris suum eggs to elucidate the effect of the individual reagents and relevant combination of reagents on the eggs. The percentages of viable eggs in samples treated with acid-alcohol alone and in combination with diethyl ether or ethyl acetate were 52, 27, and 4%, respectively, whereas in the rest of the samples the viability was about 80%. Neither the acid nor the diethyl ether alone caused any decrease in egg viability. Thus, the observed inactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution. Inactivation of the eggs was prevented by limiting the direct exposure to the extraction reagents to 30 min and diluting the residual concentration of acid-alcohol in the sample by a factor of 100 before incubation. Also, the viability of the eggs was maintained if the acid-alcohol solution was replaced with an acetoacetic buffer. None of the reagents used for the flotation step of the sample cleaning procedure (ZnSO₄, MgSO₄, and NaCl) or during incubation (0.1 N H₂SO₄ and 0.5% formalin) inactivated the Ascaris eggs under the conditions studied.     

Assessment of environmental contamination with soil-transmitted helminths life stages at school compounds,households and open markets in Jimma Town,Ethiopia

BackgroundIt remains largely unknown where and how infections with soil-transmitted helminths (STHs; Ascaris, Trichuris, Necator and Ancylostoma) occur. We therefore aimed to identify possible sources of infection by assessing the environmental contamination in an STH-endemic area.MethodsWe first performed a series of laboratory experiments designed to optimize a soil straining-flotation method to detect and quantify Ascaris and Trichuris eggs in soil, and to validate the diagnostic performance of the optimized method when followed by microscopy and qPCR. In a second phase, we applied this method to assess the level of STH contamination in 399 environmental samples collected from 10 school compounds, 50 households and 9 open markets in Jimma Town (Ethiopia). Subsequently, we explored associations between the environmental contamination and both the corresponding STH epidemiology at the level of the schools and the household characteristics. Finally, we assessed the knowledge, attitude and practice (KAP) towards STHs in school children.Principal findingsOur soil straining-flotation method has an analytical sensitivity of 50 eggs per 100 grams of soil and egg recovery rate of 36.0% (Ascaris) and 8.0% (Trichuris). The analysis of field samples with both microscopy and qPCR revealed the presence of 8 different helminth species of medical importance, including but not limited to the human STHs. There was a significant association between the environmental contamination and prevalence of any STH infections at the school level only. The KAP indicated a lack of knowledge and awareness of STHs.Conclusions/SignificanceOur optimized straining-flotation method has a moderate diagnostic performance and revealed that life stages of helminths are ubiquitous in the environment, which might be due to the poor sanitary facilities at both the schools and the households, and a poor level of KAP towards STHs. Further research is required to gain more insights into the contribution of these life stages to transmission.     

Ancient Mitochondrial DNA Analyses of Ascaris Eggs Discovered in Coprolites from Joseon Tomb

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples.     

Enumeration of Salmonellae in Table Eggs,Pasteurized Egg Products,and Egg-Containing Dishes by Using Quantitative Real-Time PCR

Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (C_q) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products.     

A combined microscopic-molecular method for the diagnosis of strongylid infections in sheep

We evaluated a combined microscopic-molecular approach for the diagnosis of key strongylid infections in sheep using panels of well-defined control and test samples. The method established is based on the separation of nematode eggs from faecal samples using a salt flotation procedure, the extraction and column-purification of genomic DNA, followed by real-time PCR and melting-curve analysis. Specific and semi-quantitative amplification from (a minimum of 0.1-2.0 pg) genomic DNA of Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp., Cooperia oncophora, Oesophagostomum columbianum, Oesophagostomum venulosum or Chabertia ovina is achieved using a specific, forward oligonucleotide primer located in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) together with a conserved reverse primer in the large subunit of rDNA. Using a panel of well-defined genomic DNA samples from eggs from sheep monospecifically infected with H. contortus or Te. circumcincta, there was a correlation between cycle threshold (Ct) values in the PCR and numbers of egg per gram of faeces, thus allowing the semi-quantitation of parasite DNA in faeces. The findings of the present study indicate that a microscopic-molecular approach provides a useful tool for diagnosis, for epidemiological and ecological surveys as well as for integration into parasite monitoring, drug resistance (i.e. ‘egg count reduction’) testing or control programmes, particularly following semi- or full-automation.     

Age, distribution and abundance of viable resting eggs of Acartia pacifica (Copepoda: Calanoida) in Xiamen Bay, China

The distribution and abundance of viable resting eggs of copepod Acartia pacifica in Xiamen Bay, China, were determined in the laboratory by the presence of nauplii hatched from the sediments. Sediment cores to a depth of 30 cm, sliced at 1.0 cm intervals, showed that most viable resting eggs of A. pacifica occurred near the sediment surface (0-5 cm), and the number of viable eggs sharply decreased with depth of the sediment, although resting eggs remained viable as deep as 23 cm. ²¹⁰Pb analyses of the sediments indicated that the maximum age of viable eggs of A. pacifica was 20.5 years and the mean egg age was 4.3 years. The egg mortality of A. pacifica in the sediment was 0.1408 year⁻¹, or 85.92% annual egg survival, calculated by regressing ln(egg density) on the age of the sediment. The horizontal distribution of viable resting eggs ranged from 2.27×10³ to 3.85×10⁵ m⁻², with a mean value of 9.49×10⁴ m⁻². Regressions between viable eggs of A. pacifica and all fine-fraction particle size classes (at 2 μm intervals) were not significant. The accumulation of viable resting eggs that can persist for an extended period of time provided evidence for the existence of an egg bank of A. pacifica in the seabed of Xiamen Bay.     

Comparison of the egg recovery rates and limit of detection for soil-transmitted helminths using the Kato-Katz thick smear,faecal flotation and quantitative real-time PCR in human stool

BackgroundMonitoring the success of soil-transmitted helminth (STH) control programs relies on accurate diagnosis and quantitative assessment of infection prevalence and intensity. As preventative chemotherapeutic program coverage for STH expands, the necessity of gaining insights into the relative or comparative sensitivities, in terms of limits of detection (LOD) and egg-recovery-rates (ERR) for microscopy and quantitative polymerase chain reaction qPCR-based diagnostic techniques becomes imperative to inform suitability for their intended use for large scale STH monitoring and treatment efficacy studies.Methodology/Principal findingsThe diagnostic performance in terms of ERR and LOD of the Kato-Katz (KK) thick smear technique, sodium nitrate (NaNO₃) faecal floatation (FF) and qPCR for the accurate detection and enumeration of STH eggs were calculated and expressed in eggs per gram (EPG), by experimentally seeding parasite-free human faeces with Ascaris spp., Trichuris spp. and Necator americanus eggs representing low, medium and high intensity infections. The efficiency of NaNO₃ flotation was also calculated over a range of specific gravities (SpGr) for the optimum recovery of STH eggs. FF of SpGr 1.30 recovered 62.7%, 11% and 8.7% more Trichuris spp., Necator americanus and Ascaris spp. eggs respectively, than the recommended SpGr of 1.20. All diagnostic methods demonstrated strong direct correlation to the intensity of seeded EPG. KK and FF (SpGr 1.30) resulted in significant lower ERRs compared to qPCR (p <0.05). qPCR demonstrated significantly (p <0.05) greater sensitivity with an ability to detect as little as 5 EPG for all three STH, compared to 50 EPG by KK and FF (SpGr 1.30).Conclusions/SignificanceThis study compares the diagnostic parameters in terms of LOD and ERRs of STHs for the KK, FF and qPCR. These results indicate that the diagnostic performance of qPCR assays should be considered by control programs in the phase that aims to seek confirmation of transmission break and cessation of preventive chemotherapy in low-transmission settings, in line with the control targets of the WHO neglected tropical diseases 2030 Roadmap.     

Ascaris suum: Immunosuppression in mice during acute infection

Mice inoculated by stomach intubation with 10,000 embryonated Ascaris suum eggs, 4, 11, or 21 days before an intraperitoneal (ip) immunization with 2 × 10⁸ sheep erythrocytes (SRBC) had reduced numbers of direct (IgM) splenic hemolytic plaques measured at 4 days after immunization and only a marginal reduction in indirect plaques (IgG) measured at 9 days after immunization. Lower dosages of Ascaris eggs or simultaneous inoculation of Ascaris eggs and SRBC did not suppress antibody responses to SRBC. No reduction in a secondary antibody response to SRBC injected 4 days after Ascaris inoculation was observed. IgM and IgG hemagglutinin titers, as distinguished by 2-mercaptoethanol sensitivity, were suppressed in mice injected ip with 10⁸ SRBC 10 days following inoculation of 10,000 Ascaris eggs, but titers in both Ig classes were similar in infected and control mice injected with 2 × 10⁹ SRBC. At Day 20, antibody titers following ip injection of 1.0 or 100 μg of ovalbumin in alum were reduced in mice infected with 10,000 Ascaris eggs 4 days before antigen injection.Contact hypersensitivity to oxazalone was not altered in mice sensitized at 5 or 14 days after inoculation of 10,000 Ascaris eggs. The delayed hypersensitivity response, measured by footpad swelling, to an optimum intravenous sensitizing dosage of SRBC was inhibited in mice sensitized 10 days after Ascaris infection, but not inhibited in mice sensitized at 21 or 32 days after infection. In contrast, the delayed hypersensitivity response to subcutaneous sensitization with SRBC 10 days after Ascaris infection was not altered.     

Molecular characterization of hard tick <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis> from China by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, the entire first and second internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) of Haemaphysalis longicornis from China were amplified by polymerase chain reaction. The 45 representative amplicons were sequenced, and sequence variation in the ITS was examined. The ITS sequences of H. longicornis were 3644 bp in size, including the part of 18S rDNA, 28S rDNA sequences and the complete ITS-1, 5.8S rDNA and ITS-2 sequences. Sequence analysis revealed that the ITS-1, 5.8S rDNA and ITS-2 of this hard tick were 1582, 152, and 1610 bp in size, respectively. The intra-specific sequence variations of ITS-1 and ITS-2 within H. longicornis were 0–2 and 0–2.2%; however, the inter-specific sequence differences among members of the genus Haemaphysalis were significantly higher, being 35.1–55.2 and 37–52% for ITS-1 and ITS-2, respectively. The molecular approach employed in this study provides the foundation for further studies of the genetic variation of H. longicornis from different hosts and geographical origins in China.     

Orientobilharzia turkestanicum is a member of Schistosoma genus based on phylogenetic analysis using ribosomal DNA sequences

In the present study, samples representing Orientobilharzia turkestanicum from cattle, sheep, cashmere goat and goat in Heilongjiang Province, China, were characterized and grouped genetically by sequences of internal transcribed spacer (ITS, including ITS-1 and ITS2) and 28S ribosomal DNA (28S rDNA). The ITS and 28S rDNA were amplified by polymerase chain reaction (PCR) and then sequenced and compared with that of other members of the Schistosomatidae available in GenBank™, and phylogenetic relationships between them were re-constructed using the neighbor-joining and maximum parsimony methods. The lengths of ITS-1, ITS-2 and 28S rDNA sequences for all O. turkestanicum samples from different hosts were 384 bp, 331 bp and 1304 bp, respectively. While the ITS-1 sequences of O. turkestanicum from each of the four different hosts, and ITS-2 of O. turkestanicum from cattle, sheep and cashmere goat were identical, respectively, the ITS-2 of O. turkestanicum from goat differed from that of O. turkestanicum from cattle, sheep and cashmere goat by one nucleotide. The 28S rDNA sequences of O. turkestanicum from sheep and cashmere goat were identical, but differed from that of O. turkestanicum from cattle and goat by two nucleotides, with the latter two also having identical 28S rDNA sequence. Phylogenetic analyses based on the combined sequences of the ITS-1 and ITS-2, or the 28S rDNA sequences placed O. turkestanicum within the genus Schistosoma, and it was phylogenetically closer to the African schistosome group than to the Asian schistosome group. These results should have implications for studying the origin and evolution of O. turkestanicum and other members of the Schistosomatidae.     

ITS1 intra-individual variability of Ascaris isolates from Brazil

The zoonotic potential of Ascaris infecting pigs has stimulated studies of molecular epidemiology with internal transcribed spacer 1 (ITS1) as the target. The aim of this study was to determine the value of Ascaris ITS1 as a molecular marker through assessing the intra-individual genetic diversity of Ascaris isolates from two geographical areas of Brazil. DNA was extracted from single isolated eggs, ITS1 PCR was performed, and the PCR products were cloned and sequenced. Clone analysis showed high ITS1 intra-individual variability revealed by 2–4 ITS1 genotypes/haplotypes per sample (egg). Two genotypes, G1 and G6, and 13 new haplotypes were detected and characterized. The most prevalent in humans, G1 and/or the Brazilian G6, were detected in all samples. Except for genotype G1, no relationship was observed between Brazilian ITS1 genotypes/haplotypes and those previously described in China, Bangladesh, Japan, United Kingdom, Australia, and Denmark, with respect to geographic origin or host affiliation. However, an association between the two geographically separated Brazilian ITS1 isolates was observed. The ITS1 intra-individual variability revealed in this study indicated that the use of this genetic region to discriminate human and pig Ascaris genotypes should be reconsidered.     

Detection and Investigation of Soil Biological Activity against Meloidogyne incognita

Greenhouse experiments with two susceptible hosts of Meloidogyne incognita, a dwarf tomato and wheat, led to the identification of a soil in which the root-knot nematode population was reduced 5- to 16-fold compared to identical but pasteurized soil two months after infestation with 280 M. incognita J2/100 cm³ soil. This suppressive soil was subjected to various temperature, fumigation and dilution treatments, planted with tomato, and infested with 1,000 eggs of M. incognita/100 cm³ soil. Eight weeks after nematode infestation, distinct differences in nematode population densities were observed among the soil treatments, suggesting the suppressiveness had a biological nature. A fungal rRNA gene analysis (OFRG) performed on M. incognita egg masses collected at the end of the greenhouse experiments identified 11 fungal phylotypes, several of which exhibited associations with one or more of the nematode population density measurements (egg masses, eggs or J2). The phylotype containing rRNA genes with high sequence identity to Pochonia chlamydosporia exhibited the strongest negative associations. The negative correlation between the densities of the P. chlamydosporia genes and the nematodes was corroborated by an analysis using a P. chlamydosporia-selective qPCR assay.     

Cdc14 protein phosphatase and topoisomerase II mediate rDNA dynamics and nucleophagic degradation of nucleolar proteins after TORC1 inactivation

Nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase elicits nucleophagy degrading nucleolar proteins in budding yeast. After TORC1 inactivation, nucleolar proteins are relocated to sites proximal to the nucleus–vacuole junction (NVJ), where micronucleophagy occurs, whereas ribosomal DNA (rDNA encoding rRNA) escapes from the NVJ. Condensin-mediated rDNA condensation promotes the repositioning and nucleophagic degradation of nucleolar proteins. However, the molecular mechanism of TORC1 inactivation-induced chromosome condensation is still unknown. Here, we show that Cdc14 protein phosphatase and topoisomerase II (Topo II), which are engaged in rDNA condensation in mitosis, facilitate rDNA condensation after TORC1 inactivation. rDNA condensation after rapamycin treatment was compromised in cdc14-1 and top2-4 mutants. In addition, the repositioning of rDNA and nucleolar proteins and nucleophagic degradation of nucleolar proteins were impeded in these mutants. Furthermore, Cdc14 and Topo II were required for the survival of quiescent cells in prolonged nutrient-starved conditions. This study reveals that these factors are critical for starvation responses.     

Bacterial rRNA Genes Associated with Soil Suppressiveness against the Plant-Parasitic Nematode Heterodera schachtii

The goal of this study was to identify bacteria involved in soil suppressiveness against the plant-parasitic nematode Heterodera schachtii. Since H. schachtii cysts isolated from the suppressive soil can transfer this beneficial property to nonsuppressive soils, analysis of the cyst-associated microorganisms should lead to the identification of the causal organisms. Our experimental approach was to identify bacterial rRNA genes (rDNA) associated with H. schachtii cysts obtained from soil mixtures with various levels of suppressiveness. We hypothesized that we would be able to identify bacteria involved in the suppressiveness by correlating population shifts with differing levels of suppressiveness. Soil treatments containing different amounts of suppressive and fumigation-induced nonsuppressive soils exhibited various levels of suppressiveness after two nematode generations. The 10%-suppressive-soil treatment contained numbers of eggs per gram of soil similar to those of the 100%-suppressive-soil treatment, indicating that the suppressive factor(s) had been transferred. Bacterial rDNA associated with H. schachtii cysts were identified using a culture-independent method termed oligonucleotide fingerprinting of rRNA genes. Bacteria from five major taxonomic groups (Actinobacteria, Cytophaga-Flexibacter-Bacteroides, α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria) were identified. Three bacterial rDNA groups contained clones that were more prevalent in the highly suppressive soil treatments than in the less suppressive treatments, indicating a potential involvement in the H. schachtii suppressiveness. When these three groups were examined with specific PCR analyses performed on H. schachtii cysts that developed in soils treated with three biocidal compounds, only one bacterial rDNA group with moderate to high sequence identity to rDNA from several Rhizobium species and uncultured α-proteobacterial clones was consistently associated with the highly suppressive treatments. A quantitative PCR analysis confirmed the association of this Rhizobium-like rDNA group with the H. schachtii suppressiveness.     

Primary structure of the 5.8S gene of rRNA and internal transcribed spacers of rDNA in diploid wheat Triticum urartu thum. ex. gandil

Nucleotide sequences of 5.8S rRNA gene and rDNA internal transcribed spacers ITS-1 and ITS-2 were determined in diploid wheat Triticum urartu. It was shown that 5.8S rRNA gene of this wheat species consists of 163 base pairs and GC-content is 59.5%. When comparing 5.8S rRNA sequences in diploid wheat, rice and lupine and also 5.8S rRNA in hexaploid wheat and horse beans a high evolutional conservatism of its structure was revealed. The size of ITS-1 and ITS-2 in Tr. urartu is 219 and 225 base pairs long correspondingly. While comparing structures of similar rDNA regions of Tr. urartu, rice and maize a high level of homology was found only between nucleotides adjoining genes of high molecular rRNAs. In ITS-1 of Tr. urartu an insertion of 5'-GACGACGACATTGTCCGTC-3' was found, which is absent in maize and rice.     

Ascaris and Escherichia coli Inactivation in an Ecological Sanitation System in Port-au-Prince,Haiti

The goal of this study was to evaluate the microbial die-off in a latrine waste composting system in Port-au-Prince, Haiti. Temperature data and samples were collected from compost aged 0 – 12+ months. Samples collected from compost bin centers and corners at two depths were assessed for moisture content, E. coli concentration, and Ascaris spp. viability. Center temperatures in compost bins were all above 58 °C, while corner temperatures were 10 – 20 °C lower. Moisture content was 67 ± 10% in all except the oldest compost. A 4-log reduction in E. coli was observed over the first sixteen weeks of composting at both locations and depths, after which E. coli was undetectable (LOD: 142 MPN g^-1 dry weight). In new compost, 10.4% and 8.3% of Ascaris eggs were viable and fully embryonated, respectively. Percent viability dropped to zero in samples older than six weeks. These findings indicate that the Haitian EcoSan composting process was effective in inactivating E. coli and Ascaris spp. in latrine waste within sixteen weeks. This study is one of the first to document efficacy of an ecological sanitation system under field conditions and provides insight into composting methods and monitoring for other international settings.