Viable Ascaris Research Articles

Investigating helminth eggs and Salmonella sp. in stabilization ponds treating septage

Sludge management arises as a relevant problem after being accumulated in primary ponds of septage treatment plants. One of the most attractive options for sludge disposal is its use in agriculture and then specific guidelines regarding hygienic quality must be fulfilled. This study aimed at evaluating the storage time needed to inactivate Ascaris eggs and Salmonella in sludge accumulated in a primary pond treating septage. Raw septage exhibited very low concentrations of viable Ascaris eggs, thus experiments with Ascaris suum eggs spiking were conducted. The concentration of Ascaris eggs in the solids accumulated at the bottom of the pond was 20 eggs/g of total solids (g TS) at the time of pond closure. Although it decreased, some eggs remained viable (0.59 mean viable eggs/g TS) up to 20 months of in-pond storage of the biosolids. Salmonella survival was studied after developing an analytical method that inhibited the native flora. Sludge was seeded with Salmonella enteritidis. An equation adequately describing Salmonella die-off in biosolids subjected to 115 days of in-pond storage/dewatering, was found to be represented by the regression: y = log MPN Salmonella/g TS = 6.67 x t(-0.086), with t = storage time elapsed in days. The initial concentration was 7.0 x 10(6) MPN/g TS and the removal efficiency was 99%.

Liming as an advanced treatment for sludge sanitisation: helminth eggs elimination— Ascaris eggs as model

The presence of helminth eggs ( Ascaris eggs) in sewage sludge may constitute a sanitary risk when used as agricultural fertiliser. Sanitisation of sewage sludge can be achieved by treatment with quick lime, a process that destroys sludge pathogens in two ways: pH increase and temperature rise. Among the pathogens of epidemiological relevance, Ascaris eggs are the most resistant to liming, and, hence, may serve as indicators of hygienic quality of biosolids. This research aims at defining, between 50°C and 60°C, the time required in the case of limed sludge to obtain a product with a negligible level of viable Ascaris eggs. To achieve this objective, investigations on inactivation kinetics of Ascaris eggs were conducted in the following products: contaminated milk of lime; naturally contaminated sludge treated with slaked lime and heat; naturally contaminated sludge treated with quick lime; and sludge treated at full scale with quick lime. For the inactivation kinetics where a negligible level of Ascaris eggs was reached, the inactivation threshold was determined. Depending on the experimental situation, the inactivation threshold period was found to fluctuate between 5 and 75 min at 55°C and between 1 and 8 min at 60°C.

The potential for disinfection of separated faecal matter by urea and by peracetic acid for hygienic nutrient recycling

No efficient, reliable, and scale independent disinfection methods for toilet waste are available today for safe recycling of plant nutrients. Therefore, two chemical treatment methods, addition of urea or of PAA (a quaternary mixture of 15% peracetic acid, 15% hydrogen peroxide and 30% acetic acid), were evaluated for disinfection of faecal matter. Degradation of the added urea resulted in 30 g of ammonia nitrogen per kilogram of treated matter and a pH increase to approximately 9.3. This produced an efficient disinfection of E. coli, Enterococcus spp., and Salmonella spp. within 3 weeks (>6log 10 reduction) and a reduction of the chemical resistant Salmonella typhimurium 28b phage, corresponding to a decimal reduction within 7.5 days. No viable Ascaris suum eggs were found after 50 days of treatment. No reduction of spore forming Clostridia spp. was observed. Urea treatment proved to be efficient for disinfection of source separated faecal matter in a scale independent method used for safe recycling of nutrients found in the faecal matter. PAA reduced all of the above indicator organisms within 12 h after application. For this faecal material, with a dry matter content of approximately 10%, an addition of 0.5–1% of PAA (active substance, corresponding to 3.3–6.7% of the Proxitane ® 15 used) was required before no viable organisms were found in the material. However, this was not tested for the A. suum. No viable spore-forming bacteria or phages were detected. A high rate of bacteria regrowth occurred at 0.15% dosage and 5 days of treatment. PAA is an efficient alternative for disinfection of separated faeces if a rapid treatment is needed.

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 H(2)SO(4)) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-microm-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 (ZnSO(4), MgSO(4), and NaCl) or during incubation (0.1 N H(2)SO(4) and 0.5% formalin) inactivated the Ascaris eggs under the conditions studied.

Monitoring of parasites in domestic sewage

The evaluation of the presence of parasites in semisolid and liquid sewage in Argentinian Patagonia in view of the fact that this is a restriction for its use. The samples taken at 4 Domestic Sewage Plants were analyzed in accordance with Standard Methods for the Examination of Water and Wastewater, Environmental Protection Agency, World Health Organization and some other classifications. Only 2 of 6 semisolid samples analyzed had non-viable Ascaris lumbricoides eggs. Of the 10 liquid samples analyzed, only 2 did not contain eggs whereas the remaining ones had pathogens of categories I (Giardia sp., Hymenolepis nana and Enterobius vermicularis) and III (Ascaris lumbricoides, Ancylostoma duodenale and Trichuris trichiura). All semisolid samples turned out to be satisfactory for use as fertilizer as no viable Ascaris lumbricoides eggs were found in any of them. But only 6 of the liquid samples were satisfactory for use as they had no eggs or their concentrations was equal to or less than 1 egg per litre.

Survival of Salmonellas and Ascaris Suum Eggs in a Thermophilic Biogas Plant

In a continuous biogas plant, receiving manure from 200 dairy cows and 400 calves and young stock, survival of salmonellas and Ascaris suum eggs was studied. The bacteria and parasite eggs were kept in filter sacs in the manure that had a temperature of 55 degrees C. No viable salmonellas or Ascaris suum eggs could be found after 24h in the digester. Survival of salmonellas and Ascaris suum eggs was also studied in the manure pit where the manure was stored after digestion. The temperature in the manure pit varied between 22-27 degrees C. Salmonellas survived 35 but not 42 days. On day 56, when the experiments had to be stopped, 60% of the Ascaris eggs were viable.

Identification of the major Ascaris allergen and its purification to homogeneity by high-performance liquid chromatography

The major allergen from the body fluid of adult Ascaris suum (ABF) has been identified and purified to homogeneity using gel permeation high-performance liquid chromatography (HPLC). The purity of the Ascaris body fluid allergen (ABA-1) was confirmed by HPLC and SDS-PAGE. ABA-1 appears to be a 25-kDa dimer in its native form, which runs as a 10-kDa monomer on SDS-PAGE under reducing conditions. The allergenicity of the HPLC-purified protein was confirmed using isolated in vivo-sensitised mast cells from infected rats in an in vitro histamine release assay. ABA-1 is responsible for >80% of the allergenicity of ABF in this sensitive and specific system. Amino acid composition analysis and N-terminal amino acid sequencing were performed on ABA-1. Comparisons are made between ABA-1 and some of the heterogeneous Ascaris allergen preparations previously published. It is suggested that Asc-1 and allergen A both contain ABA-1 in large quantities and that discrepancies in the literature result from contaminating proteins in these preparations and technical differences in characterization of the predominant molecules present in the preparations. Compositional data suggests that the ABA-1 monomer is a molecule of 94 amino acids (based on a molecular weight estimate of 10 kDa) with a composition resembling that previously published for allergen A. The first 10 amino acids are identical to those of a protein affinity purified from the body fluid of Ascaris suum at the Wellcome Laboratories for Experimental Parasitology (WLEP-14K). The similarity between ABA-1 and WLEP-14k is also apparent on SDS-PAGE. ABA-1 appears also to be the major protein component in the body fluid of a viable Ascaris lumbricoides isolated from a hospital patient.

Helminth eggs in marine and river sediments

This investigation examines the sources of parasite contamination and their effect on the contamination of three types of marine sediments of a specific coastal region: marine sediments, river sediments, and sewage sludge. In the sludge, concentrations varied between 2 × 10 1 to 3.4 × 10 2 eggs kg −1 with 30% of samples containing at least four different types of eggs (nematode and cestode eggs). In the river sediment, helminths eggs were found in 90% of samples (sector Besos) and in 80% of samples (sector Llobregat). Only 20% of the littoral marine sediments from the Besos were positive for helminth eggs but in the sludge effluent zone all the samples were positive. At 7000 m offshore no samples contained helminth eggs. In the Llobregat sector, 35% of samples contained helminth eggs; there was no apparent difference in the degree of parasite contamination between the littoral (200 m offshore) and the intermediate zones (1000 m offshore). At 4000 and 12 000 m offshore no samples contained helminth eggs. The viability of Ascaris eggs was studied. Globally the percentage of viable eggs was always higher in Besos sector than in Llobregat. If all sediment river samples contained viable eggs we have recognized in some sediment marine sample viable Ascaris eggs: in Besos sector 14% of viable Ascaris eggs were still found at 3000 m and 4000 m offshore while only samples collected from littoral zone in Llobregat sector contained 5% of viable Ascaris eggs.

The protective effect of viable and nonviable Ascaris suum larvae and egg preparations in mice.

Summary The protective properties of various second-stage larval and egg preparations of Ascaris suum were evaluated in mice. Groups of mice were injected by several “immunization” schedules with (1) lyophilized and live embryonated eggs, (2) lyophilized and live hatched second-stage larvae, (3) supernate of ground embryonated eggs, (4) live embryonated eggs per os and (5) excretions and secretions of second-stage larvae. Immunization also was attempted by planting diffusion chambers containing live second-stage larvae intraperitoneally in mice. The criterion of immunity was a significantly lower number of larvae in the lungs of mice 8 days after a challenge infection by embryonated eggs by mouth. The results show that under the conditions of the experiment, a marked protective immunity was produced when live larvae were used for the immunization. Immunization with excretions and secretions of second-stage larvae resulted in a significant immunity, as did implanting diffusion chambers containing second-stage larvae. However, the degree of resistance produced by these latter two methods was not as high as that produced by actual infection.

The Effects of Different Atmospheres and Various Supplements on the In vitro Survival and Growth of Ascaris suum Larvae

The in vitro cultivation of Ascaris larvae was carried out by supplementing either Eagle's medium or medium 199 with 20% calf or bovine serum. Growth was observed only when an initial concentration of 20,000 or more larvae/10 ml of medium were cultured at 37.5 C under partially anaerobic conditions. By this procedure a small percentage of the larvae reached sizes which made their isolation feasible. Continued development of the isolated larvae occurred only after the normal atmosphere was replaced by one of a nitrogen-5% CO2 mixture. Larvae were maintained for as long as 110 days and reached sizes up to 2,300 by 83 ,. In a previous paper (Cleeland and Laurence, 1962) a simple method for hatching large numbers of viable Ascaris larvae was described and the cultivation of these larvae in vitro in medium 199 (Morgan et al., 1950) supplemented with 20 to 30% bovine serum was reported. Survival for periods of 60 days at 37 C and 100 days at room temperature was observed along with growth of the larvae (up to 600 by 30 4) at the higher temperature when cultivation was carried out under partially anaerobic conditions. It has subsequently been found possible to increase both the time of survival and growth of the larvae. The present paper reports the results of these studies along with the effect of different atmospheric environments on their development and the initial attempts to define their nutritional requirements. MATERIALS AND METHODS Collection and preparation of infective eggs The collection and preparation of infective eggs were carried out as previously described (Cleeland and Laurence, 1962). However, most of the eggs used in the present study were obtained from the posterior two-thirds of the uteri of female worms, and a majority of these eggs never developed to the vermiform embryonic state. Consequently, to obtain enough larvae for study and eliminate the large amount of contaminating egg material, the following procedure was adopted. Hatching and preparation of the larval inoculum The egg suspensions containing approximately 10 to 20% infective eggs were washed several times with 0.01 M phosphate buffered saline, then resuspended in 20 ml of medium 199 to a concentration of 10,000 to 20,000 eggs/ml and transferred to a 125-ml Erlenmeyer flask. Hatching was carried out as reported earlier (Cleeland and Laurence, 1962) with a magnetic stirrer for a period of 24 hr at 22 to 25 C. Over 90% of the infective and a majority of the noninfective eggs were ruptured by this treatment, and most of the free larvae appeared to be undamaged and viable. The contents of two or three flasks were combined, centrifuged at 200 g for 10 min, and the supernatant fluid removed. The sediment was resuspended in 10 ml of fresh medium 199 and transferred to a 15-ml centrifuge tube. After centrifugation at 200 g for 10 min, the supernate was decanted leaving a sediment consisting of two distinct layers. The careful addition of 1 ml of 199 followed by gentle agitation of the tube caused most of the fluffy white upper layer to resuspend in the medium leaving the light brown lower layer intact. The resuspended material was decanted and the process repeated until only the brown layer remained. This layer, consisting mainly of live hatched larvae free of much of the eg material and all of the shell debris, was resuspended in 199 at room temperature until used (never more than 4 to 6 hr after hatching). Examination of the material in the white fluffy layer revealed that it consisted of damaged larvae, eggshells, egg debris, and from 20 to 40% of the viable larvae, depending on how carefully separation was carried out.