Inactivating Effects of Common Laboratory Disinfectants, Fixatives, and Temperatures on the Eggs of Soil Transmitted Helminths.

Soil-transmitted helminths (STH) are important and widespread intestinal pathogens of humans and animals. It is presently unknown which inactivating procedures may be universally effective for safe transport, preservation, and disinfection of STH-contaminated specimens, and this lack of knowledge may expose laboratory staff to higher risk of laboratory-acquired infections (LAI's). There are limited data on the efficacy of commonly used disinfectants and fecal fixatives for inactivating the eggs of STH. This work tested five disinfectants for surface cleanup, four storage temperature conditions, and six transport/storage fixatives, to inactivate eggs of three species of STH of animal origin (Ascaris suum "roundworm," Trichuris vulpis "whipworm" and Ancylostoma caninum "hookworm") as surrogates for human STH. Among disinfectants, exposure to 10% povidone-iodine for ≥5 min inactivated 100% of the three species tested, while 5 min exposure to 95% ethanol inactivated T. vulpis and A. caninum eggs. All of the fixatives tested had inactivation effects on A. caninum hookworm eggs within 24 h of exposure, except potassium dichromate, which required 48 h. 95% ethanol for ≥48 h inactivated eggs from all three STH species. Freezing at ≤-20°C for ≥24 h inactivated eggs of T. vulpis and A. caninum, but only freezing at -80°C for ≥24 h inactivated >99% eggs, including A. suum. This work provides an evidence base for health and safety guidelines and mitigation strategies for the handling, storage, and disposal of stool samples containing STH eggs in laboratory, health care, childcare, or veterinary settings. IMPORTANCE This study systematically evaluates common laboratory disinfectants and storage conditions for their effectiveness in inactivating the infective stages of soil-transmitted helminths (STH). Animal-infecting proxy species were chosen to represent three major groups of STH that infect humans: roundworms, whipworms, and hookworms. Previously published work in this area typically focuses on a particular inactivation method, either for a single STH species, or on a subset of closely related species. Because prediagnostic fecal specimens must be regarded as potentially infectious with a mix of species, such information may be of limited utility in a working laboratory. We provide a straightforward summary of storage and disinfection methods that can achieve complete inactivation across a range of STH species, which represents a significant advance for clinical, veterinary and research laboratory biosafety.

Transcription in pronuclei and one- to four-cell embryos drives early development in a nematode.

BACKGROUND: A long-standing view of development is that transcription is silenced in the oocyte until early divisions in the embryo. The point at which major transcription is reactivated varies between organisms, but is usually after the two-cell stage. However, this model may not be universal. RESULTS: We used RNA-seq and exploited the protracted development of the parasitic nematode Ascaris suum to provide a comprehensive time course of mRNA expression, degradation, and translation during early development. Surprisingly, we find that ∼4,000 genes are transcribed prior to pronuclear fusion and in the one- to four-cell embryos. Intriguingly, we do not detect maternal contribution of many orthologs of maternal C. elegans mRNAs, but instead find that these are newly transcribed in the A. suum zygote prior to pronuclear fusion. Ribosome profiling demonstrates that, in general, early embryonic mRNAs are not stored for subsequent translation, but are directly translated after their synthesis. The role of maternally contributed and zygotically transcribed genes differs between the nematodes A. suum and C. elegans despite the fact that the two nematodes appear to exhibit highly similar morphological patterns during early development. CONCLUSIONS: Our study indicates that major transcription can occur immediately after fertilization and prior to pronuclear fusion in metazoa, suggesting that newly transcribed genes appear to drive A. suum early development. Furthermore, the mechanisms used for controlling the timing of the expression of key conserved genes has been altered between the two nematodes, illustrating significant plasticity in the regulatory networks that play important roles in developmental outcomes in nematodes.

Silencing of germline-expressed genes by DNA elimination in somatic cells.

Chromatin diminution is the programmed elimination of specific DNA sequences during development. It occurs in diverse species, but the function(s) of diminution and the specificity of sequence loss remain largely unknown. Diminution in the nematode Ascaris suum occurs during early embryonic cleavages and leads to the loss of germline genome sequences and the formation of a distinct genome in somatic cells. We found that ∼43 Mb (∼13%) of genome sequence is eliminated in A. suum somatic cells, including ∼12.7 Mb of unique sequence. The eliminated sequences and location of the DNA breaks are the same in all somatic lineages from a single individual and between different individuals. At least 685 genes are eliminated. These genes are preferentially expressed in the germline and during early embryogenesis. We propose that diminution is a mechanism of germline gene regulation that specifically removes a large number of genes involved in gametogenesis and early embryogenesis.

Effect of temperature on embryonation of Ascaris suum eggs in an environmental chamber.

The influence of temperature on the development and embryonation of Ascaris suum eggs was studied using coarse sand medium in an environmental chamber with 50% humidity. The time required for development and embryonation of eggs was examined under 3 different temperature conditions, 5°C, 25°C, and 35°C. A. suum eggs did not develop over 1 month at the temperature of 5°C. However, other temperature conditions, 25°C and 35°C, induced egg development to the 8-cell-stage at days 5-6 after incubation. All eggs examined developed to the 8-cell stage at day 6 after incubation in the sand medium at 25°C. The higher temperature, 35°C, slightly accelerated the A. suum egg development compared to 25°C, and the development to the 8-cell stage occurred within day 5 after incubation. The formation of larvae in A. suum eggs at temperatures of 35° and 25°C appeared at days 17 and 19 after incubation, respectively. These findings show that 35° condition shortens the time for the development of A. suum eggs to the 8-cell-stage in comparison to 25°C, and suggest the possibility of accelerated transmission of this parasite, resulting from global warming and ecosystem changes.

Synthesis of N²-modified 7-methylguanosine 5'-monophosphates as nematode translation inhibitors.

Preparative scale synthesis of 14 new N(2)-modified mononucleotide 5' mRNA cap analogues was achieved. The key step involved use of an S(N)Ar reaction with protected 2-fluoro inosine and various primary and secondary amines. The derivatives were tested in a parasitic nematode, Ascaris suum, cell-free system as translation inhibitors. The most effective compound with IC(50) ∼0.9µM was a N(2)-p-metoxybenzyl-7-methylguanosine-5'-monophosphate 35.

Effects of kimchi extract and temperature on embryostasis of Ascaris suum eggs.

To determine the effects of kimchi extracts at different temperatures on larval development, Ascaris suum eggs were mixed with soluble part of 7 different brands of commercially available kimchi and preserved at either 5℃ or 25℃ for up to 60 days. A. suum eggs incubated at 25℃ showed marked differences in larval development between kimchi extract and control group. While all eggs in the control group completed embryonation by day 21, only 30% of the eggs in the kimchi extract group became embryonated by day 36 and about 25% never became larvated even at day 60. At 5℃, however, none of the eggs showed larval development regardless of the incubation period or type of mixture group. To determine the survival rate of A. suum eggs that showed no embryonation after being preserved at 5℃, eggs preserved in kimchi extracts for 14, 28, and 60 at 5℃ were re-incubated at 25℃ for 3 weeks in distilled water. While all eggs in the control group became larvated, eggs in the kimchi extract group showed differences in their embryonation rates by the incubation period; 87.4 % and 41.7% of the eggs became embryonated after being refrigerated for 14 days and 28 days, respectively. When refrigerated for 60 days, however, no eggs mixed in kimchi extract showed larval development. Our results indicate that embryogenesis of A. suum eggs in kimchi extract was affected by duration of refrigeration, and that all eggs stopped larval development completely in kimchi kept at 5℃ for up to 60 days.

Morphological changes of Ascaris spp. eggs during their development outside the host.

Information on the infective stage of Ascaris lumbricoides and the pathology caused by the parasite is widely available in the literature. However, information about early embryonic development of A. lumbricoides and its life cycle outside the host is limited. The purpose of this study was to describe the morphological changes within the developing embryo during incubation in vitro at 28 C, as well as to explore differences in egg viability during incubation. Ascaris suum eggs (4,000 eggs/ml), used as a model for A. lumbricoides , were placed for incubation in 0.1N H(2)SO(4) at 28 C in the dark for 21 days. Every day, sub-samples of approximately 100 A. suum eggs were taken from the incubation solution for microscopic evaluation. Development, morphological changes, and viability of the first 40 eggs were observed and documented with photos. During this study, 12 stages were identified in the developing embryo by standard microscopy, 2 of which had not been previously reported. By the end of the first wk, most developing embryos observed were in the late-morula stage (72.5%). On day 14 of incubation, 90% had developed to larva-1 stage, and by day 21, 100% had developed to larva-2 stage. No significant differences were found in the viability recorded in a continuum from day 5 to day 21 of incubation (chi-square, P > 0.05). The result of this study complements and expands the stages of development of Ascaris spp. outside the host previously reported in the literature. It also suggests the potential use of early stages of development of the nematode to determine viability and safety of sewage sludge, wastewater, or compost after treatment recommended by USEPA.

Effect of Temperature on Embryonation of Ascaris suum Eggs in an Environmental Chamber

The influence of temperature on the development and embryonation of Ascaris suum eggs was studied using coarse sand medium in an environmental chamber with 50% humidity. The time required for development and embryonation of eggs was examined under 3 different temperature conditions, 5degrees C, 25degrees C, and 35degrees C. A. suum eggs did not develop over 1 month at the temperature of 5degrees C. However, other temperature conditions, 25degrees C and 35degrees C, induced egg development to the 8-cell-stage at days 5-6 after incubation. All eggs examined developed to the 8-cell stage at day 6 after incubation in the sand medium at 25degrees C. The higher temperature, 35degrees C, slightly accelerated the A. suum egg development compared to 25degrees C, and the development to the 8-cell stage occurred within day 5 after incubation. The formation of larvae in A. suum eggs at temperatures of 35degrees C and 25degrees C appeared at days 17 and 19 after incubation, respectively. These findings show that 35degrees C condition shortens the time for the development of A. suum eggs to the 8-cell-stage in comparison to 25degrees C, and suggest the possibility of accelerated transmission of this parasite, resulting from global warming and ecosystem changes.

High pressure processing treatment prevents embryonation of eggs of Trichuris vulpis and Ascaris suum and induces delay in development of eggs.

High hydrostatic pressure processing (HPP) is an effective non-thermal treatment used to inactivate pathogens from a variety of food and food products. It has been extensively examined using prokaryotic organisms and protozoan's but has had limited study on metazoans. Treatment using HPP has been shown to be effective in inactivating nematode larvae in food and preventing embryonation of Ascaris suum eggs. We conducted experiments using eggs of the canine whipworm Trichuris vulpis collected from naturally infected dogs and A. suum eggs from naturally infected pigs. We observed a delay in development of eggs of T. vulpis in a preliminary experiment and conducted 2 experiments to test the hypothesis that appropriate HPP levels can induce a delay in embryonation of nematode eggs. In experiment 1, nonembryonated T. vulpis eggs in tap water were packaged in sealable bags and exposed to 138-600 megapascals (MPa; 1 MPa=10 atm=147 psi) for 60s in a commercial HPP unit. In a second experiment, nonembryonated eggs of A. suum were exposed to 138-600 MPa and treated for 60s in the same commercial HPP unit. Embyronation of T. vulpis eggs was delayed by 4 and 5 days for eggs treated with 207 and 241 MPa but eventually eggs developed and the numbers of embryonated eggs was similar to controls on day 55. Embryonation of T. vulpis eggs treated with 345 or 350 MPa was delayed by 9 days and never reached more than 5% of eggs embryonated. On day 55 post treatment, 95% of control nontreated T. vulpis eggs were embryonated, 100-65% of eggs treated with 138-276 MPa were embryonated, a maximum of 5% of eggs treated with 345-350 MPa were embryonated, and 0% of eggs treated with ≥ 400 MPa were embryonated. T. vulpis eggs treated with ≥ 400 MPa did not undergo cell division. Embryrnation of A. suum eggs was delayed by 4, 10, and 16 days for eggs treated with 207, 241, and 250MPa, respectively, compared to nontreated control eggs. A. suum eggs treated with 207 MPa eventually embryonated to similar % embryonation values as controls and 138 MPa treated eggs but eggs treated with 241 or 250 MPa were always <5% embryonated. A. suum eggs treated with ≥ 300 MPa did not undergo cell division. On the final day of examination at day 56 after treatment, the % of embryonated eggs was 92% nontreated controls, 94% treated with 138 MPa, 84% treated with 207 MPa, 2% treated with 241 or 250 MPa, and 0% treated with 276, 200, 345, 400, or 414 MPa, respectively.

Effects of high hydrostatic pressure on embryonation of Ascaris suum eggs.

High hydrostatic pressure processing (HPP) has been shown to be an effective non-thermal means of inactivating microorganisms from various food products. Little information is available regarding the effects of HPP on metazoan parasites. Outbreaks of food-borne disease have been associated with importation of food contaminated with fecal material. Ascaris suum is used as a surrogate model metazoan parasite for the human roundworm, Ascaris lumbricoides, to study the effects of treatments on the inactivation of eggs in sludge. The present study was conducted to determine the effects of HPP on A. suum eggs. Unembryonated A. suum eggs were subjected to 138-552 megapascals (MPa) for 10-60s in a commercial HPP unit. Embryonation was induced after HPP treatments by incubating eggs in 0.01N sulfuric acid at room temperature. After 21 days, 100 eggs were examined per treatment using a light microscope and the percent of embryonated eggs was determined. Embryonation was induced in 38-76% eggs that were subjected to 138 and 270MPa. No embryonation was observed in eggs exposed to pressures of 241MPa or more for 60s or in eggs exposed to 276MPa for 10-30s. These results indicate that HPP treatment could be used to protect contaminated food items by inactivating A. suum eggs and may also have potential in reducing food-borne illness resulting from fecal contamination.

[The influence of the Metarhizium fungi on the embryonic development of Ascaris suum]. / Wplyw grzybów z rodzaju Metarhizium na rozwój embrionalny Ascaris suum.

PURPOSE: The present study focused on the effect of two fungal species Metarhizium flavoviride and Metarhizium anisopliae on the embryonic development and viability of Ascaris suum larvae. RESULTS: Microscope examination revealed that the presence of the mycelium inhibited the egg development, compared to control. The fungus-exposed eggs featured zygote vacuolisation, irregular blastomere cleavage, and morphological disturbances in the stages of blastula, gastrula, and larva. The embryopathies were significantly more frequent in the eggs incubated with M. flavoviride. Also the mortality of the developed larvae was significantly higher after 60-day culture with M. flavoviride.

Fertilization state of Ascaris suum determined by electrorotation.

Electrorotation is a non-invasive technique that is capable of detecting changes in the morphology and physicochemical properties of microorganisms. The first detailed electrorotation study of the egg (ovum) of a parasitic nematode, namely Ascaris suum is described to show that electrorotation can rapidly differentiate between fertilized and non-fertilized eggs. Support for this conclusion is by optical microscopy of egg morphology, and also from modelling of the electrorotational response. Modelling was used to determine differences in the dielectric properties of the unfertilized and fertilized eggs, and also to investigate specific differences in the spectra of fertilized eggs only, potentially reflecting embryogenesis. The potential of electrorotation as an investigative tool is shown, as undamaged eggs can be subjected to further non-destructive and destructive techniques, which could provide further insight into parasite biology and epidemiology.

Effect of electric power network frequency magnetic field on embryonic development of Ascaris suum (Nematoda).

Fertilised Ascaris suum eggs were subjected to an alternating electromagnetic field of frequency 50 Hz and density 2 mT for 60 days. The developing embryos in both control and experimental cultures were examined daily under a microscope. The experiment resulted in an accelerated rate of embryogenesis in the eggs incubated in the electromagnetic field, higher rates of malformed embryos as well as much higher mortality rate of L2 larvae.

Contribution of trans-splicing, 5' -leader length, cap-poly(A) synergism, and initiation factors to nematode translation in an Ascaris suum embryo cell-free system.

Trans-splicing introduces a common 5' 22-nucleotide sequence with an N-2,2,7-trimethylguanosine cap (m (2,2,7)(3)GpppG or TMG-cap) to more than 70% of transcripts in the nematodes Caenorhabditis elegans and Ascaris suum. Using an Ascaris embryo cell-free translation system, we found that the TMG-cap and spliced leader sequence synergistically collaborate to promote efficient translation, whereas addition of either a TMG-cap or spliced leader sequence alone decreased reporter activity. We cloned an A. suum embryo eIF4E homolog and demonstrate that this recombinant protein can bind m(7)G- and TMG-capped mRNAs in cross-linking assays and that binding is enhanced by eIF4G. Both the cap structure and the spliced leader (SL) sequence affect levels of A. suum eIF4E cross-linking to mRNA. Furthermore, the differential binding of eIF4E to a TMG-cap and to trans-spliced and non-trans-spliced RNAs is commensurate with the translational activity of reporter RNAs observed in the cell-free extract. Together, these binding data and translation assays with competitor cap analogs suggest that A. suum eIF4E-3 activity may be sufficient to mediate translation of both trans-spliced and non-trans-spliced mRNAs. Bioinformatic analyses demonstrate the SL sequence tends to trans-splice close to the start codon in a diversity of nematodes. This evolutionary conservation is functionally reflected in the optimal SL to AUG distance for reporter mRNA translation in the cell-free system. Therefore, trans-splicing of the SL1 leader sequence may serve at least two functions in nematodes, generation of an optimal 5'-untranslated region length and a specific sequence context (SL1) for optimal translation of trimethylguanosine capped transcripts.

The effect of low temperatures on the development of eggs of Ascaris suum Goeze, 1782.

Eggs of A. suum were kept at -10 and -20 degrees C for 12 weeks and subsequently incubated in a thermostat at 28 degrees C. During the incubation, their development was checked every 5 days. It was found that storage at low temperatures slows down their development and reduces the number of eggs successfully completing their embryogenesis.

New telomere formation during the process of chromatin diminution in Ascaris suum.

Chromatin diminution in the parasitic nematode Ascaris suum represents an interesting case of developmentally programmed DNA rearrangement in higher eukaryotes. At the molecular level, it is a rather complex event including chromosome breakage, new telomere formation and DNA degradation. Analysis of a cloned somatic telomere (pTel1) revealed that it has been newly created during the process of chromatin diminution by the addition of telomeric repeats (TTAGGC)n to a chromosomal breakage site (Müller et al., 1991). However, telomere addition does not occur at a single chromosomal locus, but at many different sites within a short chromosomal region, termed CBR1 (chromosomal breakage region 1). Here we present the cloning and the analysis of 83 different PCR amplified telomere addition sites from the region of CBR1. The lack of any obvious sequence homology shared among them argues for a telomerase-mediated healing process, rather than for a recombinational event. This hypothesis is strongly supported by the existence of 1-6 nucleotides corresponding to and being in frame with the newly added telomeric repeats at almost all of the telomere addition sites. Furthermore, we show that telomeres are not only added to the ends of the retained chromosomal portions, but also to the eliminated part of the chromosomes, which later on become degraded in the cytoplasm. This result suggests that de novo telomere formation during the process of chromatin diminution represents a non-specific process which can heal any broken DNA end.

Developmentally regulated telomerase activity is correlated with chromosomal healing during chromatin diminution in Ascaris suum.

Telomerase is the ribonucleoprotein complex responsible for the maintenance of the physical ends, or telomeres, of most eukaryotic chromosomes. In this study, telomerase activity has been identified in cell extracts from the nematode Ascaris suum. This parasitic nematode is particularly suited as a model system for the study of telomerase, because it shows the phenomenon of chromatin diminution, consisting of developmentally programmed chromosomal breakage, DNA elimination, and new telomere formation. In vitro, the A. suum telomerase is capable of efficiently recognizing and elongating nontelomeric primers with nematode-specific telomere repeats by using limited homology at the 3' end of the DNA to anneal with the putative telomerase RNA template. The activity of this enzyme is developmentally regulated, and it correlates temporally with the phenomenon of chromatin diminution. It is up-regulated during the first two rounds of embryonic cell divisions, to reach a peak in 4-cell-stage embryos, when three presomatic blastomeres prepare for chromatin diminution. The activity remains high until the beginning of gastrulation, when the last of the presomatic cells undergoes chromatin diminution, and then constantly decreases during further development. In summary, our data strongly argue for a role of this enzyme in chromosome healing during the process of chromatin diminution.

A novel UV-damaged DNA binding protein emerges during the chromatin-eliminating cleavage period in Ascaris suum.

During the early cleavage period of Ascaris suum , chromatin diminution takes place in the somatic founder cells. In the process of chromatin diminution numerous heterochromatic blocks, consisting predominantly of highly repeated DNA, are discarded during mitotic anaphase and are later on digested in the cytoplasm. Very little is known about proteins that are involved in chromatin diminution. We have detected a nuclear protein and purified it to near homogeneity by its preferential binding to UV-damaged DNA. We termed this protein chromatin diminution associated factor 1 (CDAF1), because maximum binding activity per nucleus was observed to develop in 4-8-cell stages, when chromatin diminution occurs for the first time. CDAF1 recognizes cyclobutane pyrimidine dimers in UV-damaged double-stranded DNA. Its binding properties identify CDAF1 as a novel kind of damaged-DNA binding protein. CDAF1 activity is almost not detectable in 1-celled embryos. It increases dramatically during formation of somatic founder cells and persists up to the first larval stage. However, CDAF1 is absent in tissues of adults. These findings led us to suggest that CDAF1 plays a dual role: during the early segregative cleavage period it might be involved in chromatin diminution as a transfactor and act in nucleotide excision repair as an accessory factor throughout embryogenesis.

Stage-specific isoforms of complex II (succinate-ubiquinone oxidoreductase) in mitochondria from the parasitic nematode, Ascaris suum.

Complex II from mitochondria of the adult parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron transport observed in these organelles. In contrast, mitochondria isolated from free living second stage larvae (L2) of A. suum show much lower fumarate reductase activity than those from adults, whereas succinate dehydrogenase activities of mitochondria in both stages are comparable. In the present study, biochemical and antigenic properties of the partially purified enzymes from both larval and adult mitochondria were compared. Larval complex II eluted from the DEAE-Cellulofine column chromatography at a lower salt concentration than adult enzyme, whereas the apparent molecular size of both enzyme complexes estimated by gel permeation column chromatography was the same. The fumarate reductase activity of larval complex II was less than 3% of that of adult enzyme, and the Km values for substrates were significantly different between the two complexes. The flavoprotein subunit of larval complex II could be distinguished from that of adult complex II by two-dimensional gel electrophoresis and peptide mapping. The antibody against the smallest subunit (small subunit of cytochrome b558) of the adult enzyme did not cross-react with that of the larval enzyme. These results suggest that larval complex II differs from adult enzyme and is more similar to aerobic mammalian enzymes with low fumarate reductase activity. This is the first direct indication of the two different stage-specific forms of mitochondrial complex II.

Developmentally regulated alternative splicing of a nematode type IV collagen gene.

A comparison of the genomic DNA sequence that encodes the Ascaris suum alpha 2(IV) collagen chain with the corresponding cDNA sequence led to the identification of a putative exon that was not expressed in the cDNA. The identification of this putative exon raised the possibility that transcripts of the alpha 2(IV) gene may undergo alternative splicing. We have used a reverse transcriptase-polymerase chain reaction assay to establish that such alternative splicing does indeed occur. Our results show that the A. suum alpha 2(IV) collagen gene produces at least two similar, but not identical, transcripts via the selection of two alternative exons. Furthermore, this alternative splicing appears to be developmentally regulated, suggesting that alternative splicing may be used in order to modify the properties of type IV collagen during nematode development.