The inactivation of Ascaris suum eggs by short exposure to high temperatures

Ascaris sp. is the most prominent and resilient helminth of human health importance found in faecal sludge, making Ascaris sp. an ideal index organism for inactivation testing. Heat treatment destroys helminths,allowingfor safe handling and possible reuse of sludge. Technology developmentfocuses on rapid heating to minimize equipment size and cost. This study evaluates Ascaris suum eggs' viability with short heating time. Ascaris eggs were placed in a water bath at temperatures from 60 to 80 °C for various exposure times (5 seconds to 4 minutes) and were immediately processed and analysed via light microscopy. For all samples within these temperature and time ranges, less than 10% viable eggs were recovered. For 70, 75 and 80 °C, complete inactivation was observed for exposure time of 5 seconds and above.


INTRODUCTION
. The inability of countries to provide water, sanitation and hygiene (WASH) results in 1.3 million deaths annually due to diarrhoeal diseases. Diarrhoeal disease is the cause of one in eight child mortality cases in children under five years of age worldwide (Kotloff ).
Diarrhoea manifests generally as a symptom of bacterial and viral infections but may also be a symptom of infection by parasitic worms (helminths), with Ascaris lumbricoides of greatest concern (Brownell & Nelson ). Ascaris eggs are the most resilient of all organisms found in sludge, as they can withstand harsh environmental conditions, such as desiccation, and can survive in both aerobic and anaerobic environments for up to seven years in the soil (Pecson & Nelson ). Ascaris spp. are therefore deemed fit as index organisms for parasitic contamination, as well as overall pathogenic contamination and inactivation (Sidhu & Toze ). If a treatment process can inactivate Ascaris spp. eggs, then it is very likely all other pathogens will be destroyed as well (Maya et al. indistinguishable from one another in all stages (Daugschies et al. ). A. suum eggs were therefore used in this study.
Ventilated pit latrines (VIP) are regarded as improved sanitation by the South African government. Once full, the pits are sealed or emptied (Bhagwan et al. ). Proper disposal and treatment of human excreta is important in reducing transmission of infectious diseases. Land application of treated sludge is becoming increasingly common, thereby encouraging resource reuse and recovery, alleviating environmental contamination and reducing human health impacts (Fewtrell & Bartram ). () and Thomas et al. () reported Ascaris inactivation at 64 and 70 C, respectively, for 1 minute exposure. No studies focus on inactivation of Ascaris eggs for exposure times less than a minute (Thomas et al. ). Test parameters for this study ranged from 60 to 80 C, at exposure times of 5 seconds to 4 minutes, simulating the effects of the viscous heater during treatment. The isolated effects of heat on Ascaris eggs in water was investigated, rather than sludge, which might serve as a matrix to protect eggs against heat exposure.

MATERIALS AND METHODS
A. suum eggs were purchased from Excelsior Sentinel Inc.
(USA) and stored at 4 C until needed. Egg stock solutions were prepared from the initial egg samples with approximately 550 eggs per 1 mL of solution. A 34-litre heating water bath controlled experimental exposure temperatures. Eggs were exposed to heat treatments in 15 mL polypropylene test tubes preheated in the water bath by adding 13 mL of boiling water per tube and immersing these tubes into the water via a modified polystyrene rack. A thermocouple and data logger monitored temperature over time.
Eggs were analysed via light microscopy, before and after incubation at 25 C for 28 days in a cooling incubator.
Experimental temperatures were 60, 65, 70, 75 and 80 C, and exposure times 5, 10, 15, 30 and 45 seconds, and 1, 2, 3 and 4 minutes. For 80 C, shorter exposure times of 1-10 seconds were also tested. The water bath was preheated prior to each experiment. The thermocouple was placed into the tube prior to exposure to ensure steadystate temperature (Figure 1). For each treatment, a 1 mL aliquot of egg stock solution was placed into a 15 mL test tube and used to assess developmental states of the eggs before exposure to heat. This allowed counting of the total number of eggs per 1 mL of solution. One mL aliquots of egg stock solutions (approximately 550 eggs per 1 mL) were pipetted into the test tubes using a 1 mL Pasteur pipette and time recorded as T0. Eggs were exposed for the respective times, after which the tube was immediately removed from the water bath. Samples were returned to room temperature to avoid prolonged heat exposure, by emptying the contents of the test tube (i) into beakers containing 40-50 mL iced water at approximately 9 C or (ii) onto a 20 μm sieve within a bowl containing tap water at room temperature (Figure 1 Ammonium bicarbonate (made up with deionized water to form 99% ammonium bicarbonate, NH 4 HCO 3 by Sigma) was used to rinse the test tubes and beakers that previously contained treated samples. The tube or beaker contents were rinsed thoroughly over a 20 μm sieve. The deposit was transferred into a plastic 15 mL test tube using a Pasteur pipette and centrifuged at 1,310 × g (3,000 rpm) for 5 minutes using a bench-top centrifuge (with a swing-out rotor).
The supernatant was discarded, and the egg pellet was analysed under a light microscope at 100× and 400× magnification.
Egg viability was assessed by pipetting two drops of egg suspension onto a plain glass microscope slide and covering with a 40 × 22 mm coverslip. The eggs were categorized as either potentially viable or non-viable based on morphology.
Potentially viable (PV) eggs included those that were undeveloped (at different cell stages), embryonated with a visibly motile larva, and embryonated containing an immotile larva (Naidoo et al. ). Non-viable (NV) eggs included those that were dead (globules inside the egg shell), embryonated with a necrotic larva, and mechanically broken (Naidoo et al. ). Samples were analysed before and after incubation such that treated samples were immediately analysed, washed back into the test tube after counting, incubated for 28 days at 25 C and then re-analysed after incubation. This process was repeated for all temperatures and exposure times and for controls. Eggs were further analysed via oil immersion microscopy (1,000 × , 1,500× and 2,000×) in order to evaluate heat damage in greater detail (three-gear focusing using a magnification changer allowed for magnifications greater than 1,000×).
Normality of data was tested using a one-sample Kolmogorov-Smirnov test using IBM SPSS Statistics (version 24, IBM Corp., Armonk, NY, USA) and transformed accordingly using arcsine transformation, then analysed statistically. A nested analysis of variance (ANOVA) was run on the data using R (version 3.0.2, R Core Team 2016). The Shapiro-Wilk test was used to test for normality and a Levene's test was used to test for homogeneity of

RESULTS
Eggs that appeared undamaged and remained at the onecelled stage after treatment were scored as undeveloped pre-incubation, as embryo death could only be confirmed post-incubation. The percentage of viable eggs recovered pre-incubation is therefore much greater than post-incubation, especially for treatment combinations that did not yield visible egg damage. The p-values are not only indicators of significant differences between percentage of viable eggs recovered, but also the level of damage and the general morphological states between exposure times.
Samples exposed for 5 seconds at 70, 75 and 80 C resulted in the most significant shift in egg state, post-incubation (from undeveloped to developed) and were used as the baseline for comparison of successive exposure times (the least damage could be seen after 5 seconds). The general ANOVA (Table 1) indicated that temperature, independently, and in conjunction with secondary variables such as exposure time, processing with iced or tap water and point of analysis, had a significant effect on percentage of viable eggs recovered (p < 0.001).
The first level of nestedness compares each temperature (primary variable) against other temperatures and two controls for the percentage of viable eggs recovered after treatment. No significant difference was observed between control 1 (iced water) and control 2 (tap water) (p ¼ 0.760), indicating that the processing method had no effect on percentage viability (note: controls were not heat treated but were processed by the same method as treated samples).
There was, however, a significant difference in viable eggs recovered between each test temperature combination from 60 C to 80 C (p < 0.001), as well as between controls and heat-treated samples (p < 0.001).
The  had dropped to 0% after 4 minutes' exposure, although at 60 C, minimal development was still evident. At 60 C, percentages of viable eggs recovered post-incubation were mostly <10% per treatment, and almost 0% at 3 and 4 minutes (Figure 2(a)). Little visible egg damage was evident preincubation, thus they were initially classified as viable (undeveloped eggs). When considered together with the low viability percentages shown in Figure 2(a), successful inactivation at 60 C can be attributed to exposures of !45 seconds, which met the criterion for this study.
Significant egg viability differences were observed between 5 seconds and every time interval from 10 seconds to 4 minutes (5:10 seconds, p ¼ 0.002; 5:15 seconds up to 4 minutes, p < 0.001), indicating an increase in visible damage after 5 seconds when exposed to 65 C (Figure 2(b)).
Between exposures of 5 and 10 seconds, the percentage of viable eggs dropped to approximately 10% (post-incubation), and almost zero after 15 seconds onwards. There was no significant change in the state of eggs after treatment from 10 seconds onwards and between each time interval Figure 2 | Percentage of viable eggs recovered after treatment at 60 to 80 C for 5 seconds to 4 minutes (a)-(e), and 1 to 10 seconds (f), processed with either tap or iced water, pre-and post-incubation at 25 C for 28 days (n ¼ 3). There was no significant difference between processing method (p > 0.05), thus only iced water was used to process very short exposure samples (1 to 10 seconds) at 80 C. BI represents percentage of viable eggs recovered before incubation and AI represents percentage of viable eggs recovered after incubation.
(p > 0.05), confirming that successful inactivation ( 10% viable eggs recovered) occurred after exposures of 10 seconds and longer, meeting the criterion of this study. Complete die-off was, however, not achieved fully for either 60 or 65 C as isolated cases of egg development were evident even after 4 minutes of exposure.
When comparing across temperatures between 60 and showed that visible damage to the eggs was only apparent after exposures of 4-5 seconds (Figure 2(f)). Although eggs appeared healthy and undamaged after exposure to 80 C for 1-10 seconds, most failed to develop further after incubation. Some development was evident for eggs exposed for up to 2 seconds. Even so, development was arrested at stages of cleavage between two cells and the gastrula.
From 5 seconds and longer at 70, 75 and 80 C, little to no further development was observed in eggs post-incubation, indicating successful inactivation, meeting the 10% criterion set for this study (Figure 2(c)-2(e)).
Results from the independent samples t-test between the two processing methods (cooling by iced and tap water) indicated no significant difference in the percentage of viable eggs recovered after treatment (p > 0.05). The third level of comparison of the nested ANOVA (temperature/ exposure time/processing method) was therefore not outlined in further detail.  Complete die-off was confirmed by microscopic analysis post-incubation. with the current study, which found that eggs were able to continue development at temperatures up to 65 C when exposed to heat for up to 2 minutes (viability is visible in Figure 2(b), even if negligible). Low temperature treatment therefore requires longer exposure times.

CONCLUSIONS AND RECOMMENDATIONS
• All test temperatures (between 5 seconds and 4 minutes) met the criterion for this study, i.e., inactivation was considered successful if the recovery of viable eggs after treatment and incubation was 10% (Figure 2(a)-2(f)).
• Complete die-off within the tested exposure time range was noted for 70 C, 75 C and 80 C, however treatment at 60 C and 65 C allowed for development of a few eggs after incubation.
• Incubation of heat-treated samples is required when egg damage is not visible, in order to confirm die-off.
• The results of the current study therefore show that residence times of as low as 4 seconds at 80 C may be recommended for successful inactivation when using the viscous heater. may act as insulation for eggs exposed to heat, suggesting that higher temperatures and prolonged exposure times might be needed for testing eggs in vitro. The role of the suspension medium during heating needs to be investigated. Further work is also required to test the actual effects of heat treatment of the eggs using the viscous heater.