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℃, 25℃, and 35℃. A. suum eggs did not develop over 1 month at the temperature of 5℃. However, other temperature conditions, 25℃ and 35℃, 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℃. The higher temperature, 35℃, slightly accelerated the A. suum egg development compared to 25℃, 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℃ 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℃, and suggest the possibility of accelerated transmission of this parasite, resulting from global warming and ecosystem changes.     

Quantitative Evaluation of Viability- and Apoptosis-Related Genes in Ascaris suum Eggs under Different Culture-Temperature Conditions

Ascaris suum eggs are inactivated by composting conditions; however, it is difficult to find functional changes in heat-treated A. suum eggs. Here, unembryonated A. suum eggs were incubated at 20℃, 50℃, and 70℃ in vitro, and the gene expression levels related to viability, such as eukaryotic translation initiation factor 4E (IF4E), phosphofructokinase 1 (PFK1), and thioredoxin 1 (TRX1), and to apoptosis, such as apoptosis-inducing factor 1 (AIF1) and cell death protein 6 (CDP6), were evaluated by real-time quantitative RT-PCR. No prominent morphological alterations were noted in the eggs at 20℃ until day 10. In contrast, the eggs developed rapidly, and embryonated eggs and hatched larvae began to die, starting on day 2 at 50℃ and day 1 at 70℃. At 20℃, IF4E, PFK1, and TRX1 mRNA expression was significantly increased from days 2-4; however, AIF1 and CDP6 mRNA expression was not changed significantly. IF4E, PFK1, and TRX1 mRNA expression was markedly decreased from day 2 at 50℃ and 70℃, whereas AIF1 and CDP6 mRNA expression was significantly increased. The expressions of HSP70 and HSP90 were detected for 9-10 days at 20℃, for 3-5 days at 50℃, and for 2 days at 70℃. Taken together, incremental heat increases were associated with the rapid development of A. suum eggs, decreased expression of genes related to viability, and earlier expression of apoptosis-related genes, and finally these changes of viability- and apoptosis-related genes of A. suum eggs were associated with survival of the eggs under temperature stress.     

Effects of Disinfectants on Larval Development of Ascaris suum Eggs

The objective of this study was to evaluate the effects of several different commercial disinfectants on the embryogenic development of Ascaris suum eggs. A 1-ml aliquot of each disinfectant was mixed with approximately 40,000 decorticated or intact A. suum eggs in sterile tubes. After each treatment time (at 0.5, 1, 5, 10, 30, and 60 min), disinfectants were washed away, and egg suspensions were incubated at 25˚C in distilled water for development of larvae inside. At 3 weeks of incubation after exposure, ethanol, methanol, and chlorohexidin treatments did not affect the larval development of A. suum eggs, regardless of their concentration and treatment time. Among disinfectants tested in this study, 3% cresol, 0.2% sodium hypochlorite and 0.02% sodium hypochlorite delayed but not inactivated the embryonation of decorticated eggs at 3 weeks of incubation, because at 6 weeks of incubation, undeveloped eggs completed embryonation regardless of exposure time, except for 10% povidone iodine. When the albumin layer of A. suum eggs remained intact, however, even the 10% povidone iodine solution took at least 5 min to reasonably inactivate most eggs, but never completely kill them with even 60 min of exposure. This study demonstrated that the treatment of A. suum eggs with many commercially available disinfectants does not affect the embryonation. Although some disinfectants may delay or stop the embryonation of A. suum eggs, they can hardly kill them completely.     

Cross-reactions of sera from Toxocara canis-infected mice with Toxascaris leonina and Ascaris suum antigens.

The ELISA method using larval excretory-secretory (E/S) products and homogenized Toxocara canis, Toxascaris leonina and Ascaris suum adult worm extract were used to determine possible cross-reactions in BALB/c and C57BL/10 mice, inoculated with embryonated eggs or adult worm extract of T. canis in single and multiple doses. When we used sera of mice infected with embryonated eggs of T. canis against different heterologous antigens, we observed no cross-reactions in BALB/c mice against A. suum E/S and adult worm extract antigens with a single dose. In multiple doses this was absent too against T. leonina adult worm extract in BALB/c mice, and in both strains against A. suum E/S and adult worm extract. In BALB/c mice inoculated with adult worm extract of T. canis we did not observe cross-reactions with A. suum E/S antigen with both inoculation doses. In the remainder of the experiments, we observed cross-reactions of different intensities.     

The role of oxygen availability in the embryonation of Heterakis gallinarum eggs.

The importance of oxygen availability in the embryonation of the infective egg stages of the gastrointestinal nematode parasite Heterakis gallinarum was studied in the laboratory. Unembryonated H. gallinarum eggs were kept under either aerobic conditions by gassing with oxygen, or anaerobic conditions by gassing with the inert gas nitrogen, under a range of constant temperatures. Oxygenated eggs embryonated at a rate influenced by temperature. Conversely, eggs treated with nitrogen showed no embryonation although when these eggs were transferred from nitrogen to oxygen gas after 60 days of treatment, embryonation occurred. This demonstrated that oxygen is an essential requirement for H. gallinarum egg development, although undeveloped eggs remain viable, even after 60 days in low oxygen conditions. The effects of climate on the biology of free-living stages studied under constant laboratory conditions cannot be applied directly to the field where climatic factors exhibit daily cycles. The effect of fluctuating temperature on development was investigated by including an additional temperature group in which H. gallinarum eggs were kept under daily temperature cycles between 12 and 22°C. Cycles caused eggs to develop significantly earlier than those in the constant mean cycle temperature, 17°C, but significantly slower than those in constant 22°C suggesting that daily temperature cycles had an accelerating effect on H. gallinarum egg embryonation but did not accelerate to the higher temperature. These results suggest that daily fluctuations in temperature influence development of the free-living stages and so development cannot be accurately predicted on the basis of constant temperature culture.     

The morphogenesis of Ascaris suum to the infective third-stage larvae within the egg.

Studies of the morphology of Ascaris suum larvae developing in the egg during embryonation in vitro at room temperature showed that 2 molts take place within the egg. The first larval stage (L1) appeared in the egg after 17-22 days of cultivation, the first molt to the second larval stage (L2) took place from day 22 to day 27, and the second molt to the third larval stage (L3) started on day 27 and continued during the 60-day observation period. Infectivity of the eggs was studied by oral egg inoculation in mice and showed that the L3 are the infective stage for mice. Molting to the L3 stage occurs gradually over a period of 2-6 wk, and it is recommended to have an additional maturation period so the infectivity of an egg batch may reach maximum level.     

The role of oxygen availability in the embryonation of Heterakis gallinarum eggs

The importance of oxygen availability in the embryonation of the infective egg stages of the gastrointestinal nematode parasite Heterakis gallinarum was studied in the laboratory. Unembryonated H. gallinarum eggs were kept under either aerobic conditions by gassing with oxygen, or anaerobic conditions by gassing with the inert gas nitrogen, under a range of constant temperatures. Oxygenated eggs embryonated at a rate influenced by temperature. Conversely, eggs treated with nitrogen showed no embryonation although when these eggs were transferred from nitrogen to oxygen gas after 60 days of treatment, embryonation occurred. This demonstrated that oxygen is an essential requirement for H. gallinarum egg development, although undeveloped eggs remain viable, even after 60 days in low oxygen conditions. The effects of climate on the biology of free-living stages studied under constant laboratory conditions cannot be applied directly to the field where climatic factors exhibit daily cycles. The effect of fluctuating temperature on development was investigated by including an additional temperature group in which H. gallinarum eggs were kept under daily temperature cycles between 12 and 22°C. Cycles caused eggs to develop significantly earlier than those in the constant mean cycle temperature, 17°C, but significantly slower than those in constant 22°C suggesting that daily temperature cycles had an accelerating effect on H. gallinarum egg embryonation but did not accelerate to the higher temperature. These results suggest that daily fluctuations in temperature influence development of the free-living stages and so development cannot be accurately predicted on the basis of constant temperature culture.     

Ecdysteroids during embryonation of eggs of Ascaris suum

1. The optimal temperature for in vitro development of fertilized eggs of Ascaris suum was 24 degrees C. 2. Samples (2 X 10(7) eggs) were obtained from in vitro embryonating cultures every 3 days for 4 weeks; lipids were extracted, partially purified, fractionated with HPLC and analyzed for ecdysteroids by radioimmunoassay. 3. Free ecdysone and 20-hydroxyecdysone (20-HE) were at low levels (less than 20 pg) in freshly excised eggs and rose to maximal values on day 6 of embryonation. 4. Conjugated ecdysone and conjugated 20-HE rose to maximal values on day 9. 5. Both free and conjugated ecdysteroids were undetectable from days 15 to 27 of cultivation. 6. These profiles indicate that ecdysteroids might have a selective role in nematode embryonation and/or tanning of the egg shell.     

Embryonation and Infectivity of Ascaris suum Eggs. A Comparison of Eggs Collected from Worm Uteri with Eggs Isolated from Pig Faeces

Ascaris suum eggs were collected from pig faeces or dissected from worms obtained from the same pigs. Eggs from the two sources were allowed to embryonate in 0.1 N H2SO4, in 1 % buffered formalin or in tap water. The embryonation of the sulphuric acid and water cultures occurred at the same speed, while the formalin cultures developed slightly more slowly. By experimental inoculation of helminthfree pigs and subsequent counting of white spots in the livers and larvae in the lungs day 7 p. i., the infectivity of eggs dissected from worm uteri and embryonated in sulphuric acid (a normal laboratory procedure) was compared with that of eggs collected from faeces and embryonated in water (i.e. more naturally developed eggs). The results suggest that the two types of eggs were equally infective. For this reason the common practice of using Ascaris eggs dissected from worms for experimental infections might be acceptable.     

Development of Ascaris lumbricoides eggs from females eliminated after chemotherapy in man.

The development of Ascaris lumbricoides eggs obtained from females eliminated after treatment of infected individuals with a single oral dose of the antihelminthic drugs thiabendazole (50 mg/kg--33 patients) or levamisole (250 mg--independent of body weight--20 patients) was studied. Every female eliminated up to 72 h after treatment were dissected, the uterus isolated and sectioned into small fragments. The eggs were transferred to plastics tubes and incubated at 28 degrees C in 0.1 N H2SO4 for 100 days. Every 20 days, starting from the 20 th up to the 100 th day, the extent of egg embryonation ratio was determined. The culture of A. lumbricoides eggs obtained from females from patients treated with thiabendazole did not contain embryonated eggs until the final period of observation. In contrast, the eggs obtained from females eliminated by patients treated with levamisole (control) presented an embryonation rate of 0.0-98.0% in the same period.     

Experimental Toxocara cati infection in gerbils and rats

Mongolian gerbils and Wistar rats were inoculated orally with 240 and 2,500 Toxocara cati embryonated eggs, respectively, to evaluate the larval recovery in different tissues and organs, such as the liver, lungs, heart, kidney, and skeletal muscles after 5, 30, 49, 70, and 92 days post-infection (PI). Larval recovery rates were 1.7-30.0% in Mongolian gerbils on days 5-92 PI and 0.2-3.8% in rats on the same days. These results indicate that Mongolian gerbils and Wistar rats are suitable experimental paratenic hosts for the study of neurological toxocariasis as well as visceral toxocariasis.     

Respiratory metabolism and thiabendazole susceptibility in developing eggs of Haemonchus contortus

Weston K. M., O'Brien R. W. and Prichard R. K. 1984. Respiratory metabolism and thiabendazole susceptibility in developing eggs of Haemonchus contortus. International Journal for Parasitology14: 159–164. The respiratory metabolism of and uptake of thiabendazole (TBZ) by unembryonated (8–16 cells) and embryonated eggs of Haemonchus contortus have been compared. Lipid, which forms the greatest energy reserve in the eggs, decreases during embryonation and seems to be the sole source of respiratory energy. Trehalose increases to the same extent as glycogen decreases during this development. Isocitrate dehydrogenase (NADP⁺) and lactate dehydrogenase were not detected in the unembryonated eggs, but were present after embryonation. In addition, the activities of citrate synthase and malate dehydrogenase significantly increased during embryonation. Respiratory enzymes involving cytochrome c oxidation and reduction were detected in eggs at both stages of development. However, in line with other results indicating an increased capacity for and utilization of aerobic metabolism, the rate of oxygen uptake more than tripled during development of the eggs. Although both unembryonated and embryonated eggs took up palmitate, its metabolism to CO₂ only occurred in the embryonated eggs.The unembryonated eggs, exposed to TBZ for 4 h, concentrated it 5.9 times and the embryonated eggs 2.1 times, which are in proportion to their respective lipid contents. Uptake of TBZ was dependent on the concentration in the incubation medium and appears to be a passive process.The studies indicate that the embryonated eggs have a greater capacity for, and do utilize aerobic metabolism to a greater extent than do unembryonated eggs. The reduced susceptibility of embryonated eggs to TBZ could be associated with this metabolic difference and/or with their reduced uptake of TBZ.     

Purine nucleotide content of developing Ascaris lumbricoides eggs

Farland W. H. &; Macinnis A. J. 1978. Purine nucleotide content cf developing Ascaris lumbricoides eggs. International Journal for Parasitology8: 177–186. Populations of Ascaris lumbricoides eggs obtained from the terminal portion of the uteri of mature females were shown to develop synchronously, allowing the detection of quantitative and qualitative changes in nucleotide content during development. Application of a method for the preparation of perchloric acid-soluble fractions from Ascaris eggs utilizing Alamine 336S in chloroform is described. This method was useful in neutralizing the extract and for removal of interfering lipids.Guanine nucleotides were found in high concentration in ovarian tissue of adult female worms, and comprise more than 80% of the total acid-soluble nucleotides in 0-day eggs. Muscle tissue contained a predominance of adenine nucleotides.To determine the fate of large concentrations of guanine nucleotides present in 0-day eggs, perchloric acid-soluble fractions were prepared from embryonating eggs on various days of development. Nucleotide content was determined after fractionation on DEAE-cellulose columns,A dramatic decrease (6·2 fold) in guanine nucleotides between 5 and 8 days' embryonation was seen; [GMP] and [GDP] showed the greatest change. ATP concentration increases 3·3 fold through 21 days' embryonation. The total acid-soluble nucleotide content decreased 60% during this time. Uric acid, an end product of purine metabolism, was detectable in 5-day eggs and accumulated through the course of embryonation. The decrease of guanine nucleotides correlates temporally with the increase in DNA content during embryonation. The methodology and results of this study provide a basis for additional study of nucleic acid metabolism during development of Ascaris eggs.     

Effects of plumbagin on development of the parasitic nematodes Haemonchus contortus and Ascaris suum.

1. Plumbagin (5-hydroxy,2-methyl-1,4-napthoquinone) inhibited the motility and survival of Haemonchus contortus first-stage larvae (L1) with an ED50 of 1 microgram/ml, but was less effective in preventing the development of H. contortus to infective third-stage larvae in a faecal slurry assay. 2. Of the structural analogs tested, plumbagin was the most potent in preventing development of L1 followed in decreasing order of potency by 1,4-naphthoquinone, 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone. 3. Plumbagin had a biphasic effect on development of the fourth-stage Ascaris suum larvae that caused an increase in growth at low concentrations but was lethal at higher doses. 4. Plumbagin and 1,2-napthoquinone partially inhibited embryonation of A. suum eggs.     

Effects of immunoactivity on Ascaris suum infection in mice]

The immune response to sheep red blood cell (sRBC) was monitored in the mice infected with Ascaris suum or Trichinella spiralis. The effects of the infection with T. spiralis or the injection with cyclophosphamide(CY) as an immunosuppression agent prior to challenge infection with the embryonated eggs of A. suum were monitored in mice by means of the level of infection with A. suum and cellular and humoral immune response to sRBC. Following the oral administration of 1,000 eggs of A. suum to mice, delayed-type hypersensitivity (DTH) and rosette-forming rate were gradually decreased and reached to the lowest levels at the 5th week and 6th week postinfection, respectively, and then returned to normal at the 10th week. The hemagglutinin(HA) and hemolysin(HE) titers were gradually elevated and reached to peak at the 3rd week postinfection, and then returned to normal level. The appearance ratios of the eosinophils and mast cells were in peak at the 4th week and the 2nd week postinfection, respectively. Meanwhile the harvest ratio of A. suum larvae from the liver and lungs was 21.97% at the 1st week postinfection. Following the oral administration of 300 T. spiralis infective larvae, DTH and rosette-forming rate were gradually decreased with the lapse of time and reached the lowest values in the 30th and 21st day of postinfection, and then slightly increased and transiently decreased in the 70th and 80th day of postinfection, respectively. HA and HE titers were the lowest in the 21st and 90th day, whereas the ratios of eosinophils and mast cells were the highest on the 40th and 14th day postinfection, respectively. Following the intraperitoneal injection of CY, the body weight, the spleen weight, DTH, rosette-forming ratio, HA and HE titers, the number of WBC and the ratio of the mast cell were predominantly decreased in the 5th day, and then returned to the same value of the 1st day postinjection. The ratio of eosinophils was gradually decreased following to advance of days. At the 1st, 5th and 10th days after intraperitoneal injection of CY of 400 mg/kg, a dose with 1,000 eggs of A. suum was administered orally to mice, and harvest rate of the larvae at the 7th day postadministration was 7.07% in the 1st day, 14.94% in the 5th day, 10.1% in the 10th day, 8.02% in control group. The effect of prior infection with infective larvae of T. spiralis upon immunological sequelae of a challenge infection of mice with embryonated eggs of A. suum in 30 or 70 days interval was checked.(ABSTRACT TRUNCATED AT 400 WORDS)     

White spots of the liver in pigs experimentally infected with Ascaris suum

To make clear the relationship between Ascaris suum infection and the appearance of white spot lesions on the surface of the liver in pigs, three groups of pigs were inoculated orally with embryonated A. suum eggs and observed clinicopathologically. Group A of three pigs were inoculated 21 times with 100 eggs each of the nematode, group B of three pigs 4 times with 50,000 eggs each for 10 weeks, and group C of two pigs 2 times with 50,000 eggs each at a one-week interval. All the pigs were sacrificed at the same time 1 week after the final inoculation. Such signs of the nematode infection as dyspnea, coughing and fever appeared in all the pigs of groups B and C seven days after inoculation to continue for several days. In addition, peripheral blood eosinophilia was recorded in these animals 7 or 14 days after inoculation. At autopsy, mesh-worked white spots, some compact and others lymphonodular, were observed on the surface of the liver in all the pigs of the three groups. Main white spots were mesh-worked and lymphonodular in the pigs of group A. They were severe and compact in group B. Therefore, they were rough to the touch. In group C mesh-worked white spots fused with one another and covered the surface of the liver. These white spot lesions observed were morphologically very similar to those found in the field conditions. Complement-fixating antibodies reacting to adult A. suum antigen were detected only in sera from the pigs of group B. Moreover, antibodies involved in the intradermal reaction of immediate type were found in the pigs of groups A and B.     

萧氏松茎象的生活史、产卵和取食习性

萧氏松茎象Hylobitelus xiaoi是严重蛀害国外松的新害虫。在江西赣南地区两年发生一代,以幼虫在蛀道、成虫在蛹室或土中越冬。卵和蛹的发育起点温度分别为8.4℃和7.5℃,有效积温分别为215.9和345.3日·度。各虫态历期：13～28℃间,卵为52.9～11.4天;13～30℃间,蛹为60.1～16.4天; 25℃恒温下,幼虫128.9天。成虫靠爬行活动,极少飞翔。成虫具夜出性活动节律：即傍晚上树行取食、交配和扩散等活动,早晨回到树干基部或土缝中。成虫需取食松枝作为补充营养,产卵前期46.3天,产卵期 105.3天,卵产于近表土的寄主树皮内,每雌产卵35.7粒。幼虫5～7龄,以幼树危害最烈。     

Storage and incubation of Echinostoma revolutum eggs recovered from wild Branta canadensis, and their infectivity to Lymnaea tomentosa snails

Echinostoma revolutum eggs recovered from naturally infected wild Canada geese (Branta canadensis) were cold stored (4-6 degrees C) for up to 72 weeks. Successful hatching followed incubation for from 6 to 8 days at an optimum temperature of between 25 and 30 degrees C. A partial life cycle from adult worm to metacercarial encystment in Lymnaea tomentosa snails was completed in the laboratory. Snails were infected both by free miracidia and by ingestment of unhatched embryonated eggs. Infection was equally successful in environmental temperature ranges from 10 to 25 degrees C, and at challenge levels of 2, 5 or 10 embryonated eggs per snail. Exposure to 10 eggs was lethal. Ingestion by snails of embryonated eggs with successful infection at 10 degrees C suggests that embryonated eggs may be used to infect wild snails when the environmental water temperature has reached 10 degrees C.     

Evaluation by ELISA of anisakis simplex larval antigen purified by affinity chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of (1/4)00 and 1 microg/ml of antigenic concentration.     

Proteinase activity in Ascaris suum eggs, hatching fluid, and excretions-secretions.

Hatching fluid and the excretions and secretions (E.S.) of hatched larvae of Ascaris suum revealed proteinase activity when assayed by 2 different procedures employing collagen or casein as substrates. Both assays apparently detected similar levels of proteinase activity in hatching fluid and E.S. of hatched larvae. The Anson (casein substrate) assay worked best in 0.05 M phosphate buffer, pH 8.0. The Azocoll (collagen substrate) assay worked best in 0.05 M borate buffer at pH 8.8. Azocoll assays done at temperatures ranging from 25 to 65 C revealed maximal proteinase activity at 55 C. Analysis of hatching fluid from 18-, 21-, and 28-day-old embryos and of extracts from sonicated 0- to 28-day-old developmental stages showed that proteinase activity increased markedly 18 days after embryonation had begun. Prior to the 18th day of embryonation proteinase levels were relatively low.