Characterization of antigens of Enterobius vermicularis (pinworm) eggs

Enterobiasis (pinworm infection) caused by Enterobius vermicularis is a common parasitic infection prevalent worldwide especially in children. Infection is diagnosed by microscopic detection of E. vermicularis eggs on perianal swabs. This study aimed to characterize the antigens of E. vermicularis eggs as a preliminary step towards identifying diagnostic targets for detection in infected individuals. The study was conducted between October 2019 and February 2020, following approval from Ethics Review Committee of the Faculty of Medicine, University of Colombo (EC-19-034). E. vermicularis eggs were harvested from perianal swabs using acetone and purified with 1× PBS (pH 7.2). A portion of eggs was used for preparing antigen slides, while the rest were sonicated and vortexed with glass beads and inoculated subcutaneously (with weekly booster doses) into a Wistar rat for developing antibodies. Blood drawing from rat was done weekly for 5 weeks. Confirmation of the presence of antibodies was done by surface immunofluorescence against eggs on the antigen slides. Protein bands were determined using SDS-PAGE assay and immunogenic antigen bands were determined by reacting with antiserum after immunoblotting. The band sizes of the proteins were determined against corresponding bands of a protein ladder. Surface immunofluorescence was positive with serum obtained from day 14 post-inoculation from the Wistar rat as well as that obtained from a person with chronic enterobiasis. The most prominent and immunogenic protein bands identified from egg antigens were 21 kDa, 66 kDa, 83 kDa, 96 kDa, 112 kDa, 121 kDa, 140 kDa and 151 kDa. Methods used in this study were effective in obtaining E. vermicularis egg antigens which were immunogenic. Furthermore, surface antigens of intact eggs reacted with antibodies developed against crushed egg antigens. These findings may pave the way for the development of effective immunodiagnostics.

www.nature.com/scientificreports/ early morning prior to washing of the perianal region. Furthermore, deposition of eggs may be variable, and as such, sampling on subsequent days is essential prior to excluding infection. In addition, microscopic examination for identification of eggs requires some expertise, and can be time consuming. As such, conduct of population surveys or screening of large groups requires much planning and can be rather cumbersome. Hence, a diagnostic method which is easy to perform as well as being equally or more sensitive and specific than microscopy for the diagnosis of enterobiasis would be most useful for the control of pinworm infection. As such, this study aimed to characterize the antigens of E. vermicularis eggs as a preliminary step towards identifying those that would be useful as diagnostic targets for detection in infected individuals.

Methods
Sample collection and E. vermicularis egg isolation. Anonymized patient samples (perianal swabs) positive for Enterobius vermicularis eggs were obtained from the Department of Parasitology, Faculty of Medicine, University of Colombo. A sample of 5 ml blood was provided voluntarily by an anonymous donor diagnosed with chronic enterobiasis (pinworm infection for more than 10 years). All samples had been obtained following informed consent from the participants or their legal guardians. Ethics approval for the study was obtained from the Ethics Review Committee of the Faculty of Medicine, University of Colombo, Sri Lanka (EC-19-034), as per the guidelines published by the Forum of Ethics Review Committees of Sri Lanka 8 , and conducted in accordance between October 2019 and February 2020. Reporting in this manuscript follows the recommendations of the ARRIVE guidelines. The perianal swabs were made of 2 layers of cellophane and the bottom layer comprised a central, circular, adhesive, clearly marked, blue colour area for easy placement on the perianal skin. E. vermicularis eggs attached to these swabs were harvested by using absolute acetone, which was found to be the most effective in detaching eggs from these adhesive tapes. Five different solvents were tested initially: one fourth of the original size of the swab was cut and placed in tubes containing 1 ml of distilled water, absolute alcohol, absolute acetone, phosphate-buffered saline (PBS, pH 7.2) and potassium dichromate (K 2 Cr 2 O 7 ), and kept for 5 min at room temperature (RT), while continuously stirring with a pair of forceps. Pre-and post-egg counts were determined by examining each piece of swab under the microscope, and absolute acetone was observed to have successfully removed all eggs attached to the swab.
Extraction of eggs for the study was done thereafter by adding a cut piece of swab, one fourth of the original size of the circular central blue colour area, into 300 µl of acetone in an Eppendorf tube and kept for 1 min at RT whilst stirring with forceps. The swab was examined microscopically under × 100 magnification for any remaining eggs and was re-dipped and stirred until all eggs had been removed into the solution.
Purification of isolated E. vermicularis eggs. The eggs were purified as described below by centrifugation to remove debris, glue and acetone, followed by several steps of washing, in order to prevent any contamination/interference with antigen preparation and the production of immune sera in rats.
Several methods were tested initially to identify the most effective force for pelleting of eggs: using short spins in a mini centrifuge machine with higher centrifugal forces such as 14,000×g resulted in an egg clump which could not be reconstituted due to the sticky nature of the eggs and the presence of debris. Therefore, the eluted egg suspension was given increasing forces between 500 and 800×g (500×g, 650×g, 700×g and 800×g) with 800×g able to sediment the eggs effectively. All remaining traces of glue were removed by repetitive washing with 200 µl of acetone and finally with PBS, and then re-suspended in 250 µl of PBS.
Preparation of E. vermicularis egg antigen slides (using intact eggs). Five microlitres of the resuspended egg pellet were transferred onto a glass slide and the egg count was estimated (approximately 1000 eggs/250 µl). Final volume of the egg suspension was then adjusted to get approximately 500 eggs per 250 µl. Antigen slides were prepared with 5 μl of egg suspension per spot, containing 5-10 eggs (concentration of 1-2 eggs/μl), placed in the clean wells of a 12-well multi-spot microscope slide. Slides were air-dried overnight at RT. The eggs in each well of the slides were counted and slides were stored at − 70 °C until used.

Preparation of E. vermicularis egg antigen sample (with disrupted/crushed eggs). Egg antigens
were obtained by disrupting the egg shell and release of the larval stage within. Several egg disruption methods were tested using samples of the purified E. vermicularis egg suspension to identify the most effective method. Sonicating the egg suspension at 53 kHz with 4 mm glass beads (Marienfeld, Germany) for 30 min at RT and then vortexing for 15-30 min was successful in disrupting all eggs in the test sample.
Vortexing of the egg suspension (250 µl containing approximately 1000 eggs) for 30 min, 45 min or 1 h at repeated intervals, as well as, sonicating of the egg suspension at 53 kHz for 5, 15 or 30 min time periods at RT, resulted in intact eggs in each of the test samples. Mixing the egg suspension either with 1% Triton X-100 in PBS (v/v = 1:1) or with 2% sodium dodecyl sulfate (SDS) in PBS (v/v = 1:1) for 1 min and then vortexing for 5-15 min yielded intact eggs in the former and only partial discharge of larvae in the latter. Three repeated freeze-thaw cycles (15 min at − 80 °C followed by 10 min at 37 °C) of the egg suspension also gave intact eggs, while vortexing with glass beads for 30 min too was unsuccessful. Sonicating at 53 kHz with glass beads for 30 min at RT followed by vortexing for a similar time period finally yielded a considerable proportion of crushed eggs.
Estimation of the protein concentration of E. vermicularis egg antigen sample. Once the eggs were broken and the antigen prepared, the total protein concentration of the sample was measured using the Bradford assay 9 ; with the use of a protein standard and by plotting a calibration curve. Bovine serum albumin (BSA) was used as the protein standard, as it interfered less with the reagents used in the Bradford assay. The www.nature.com/scientificreports/ extinction coefficient of a dye-albumin complex solution is constant over a tenfold concentration range. Thus, Beer's law 10 could be applied for accurate quantification of protein by selecting an appropriate ratio of dye volume to sample concentration. The protein concentration of the test sample was determined by comparison to that of a series of protein standard (BSA) known to reproducibly exhibit a linear absorbance profile in this assay. The optical density (OD) at 595 nm wave-length was measured using a microplate reader and the calibration curve was plotted using a dilution series of BSA standard starting from 10 to 1000 μg ( Fig. 1). Then using the standard curve, the protein concentration of the sample was estimated.

Development of immune serum against E. vermicularis egg antigens. Immune serum against E.
vermicularis egg antigens was developed in a healthy young female adult Wistar rat (approximately 5-6 months old) at the rat facility of the Institute of Biochemistry, Molecular Biology and Biotechnology (IBMBB) of the University of Colombo, where it was housed in a clean polypropylene cage under the following environmental conditions: 22-23 °C temperature, 70% humidity, 12-h light and dark cycles, and with central air conditioning. Feeding was done once in two days with commercially available pelleted (rat) food and clean water (replenished daily) provided through a bottle fixed to the cage. The rat was acclimatized for a week prior to use in the study. Crude egg antigen samples, Freund's complete (FCA) and Freund's incomplete adjuvant (FIA) mixtures were made in a hood under sterile conditions. Firstly, 150 μl of egg antigen was transferred into an Eppendorf tube, to which adjuvant was added according to the immunization chart shown in Table 1, to form a total volume of 300 µl. They were mixed more than 30 cycles by sucking in and out using a 1 ml syringe with a 23 G needle and emulsified. A drop from the emulsion was put in water and observed for dispersion in order to ensure it had mixed well. The rat was mildly anaesthetized by placing in a closed desiccator containing anaesthetic ether for a few seconds and then injected with 200-250 μl of antigen-adjuvant mixture subcutaneously using a 1 ml syringe with a 23 G needle as described by Leenaars and Hendriksen 11 . The rat was placed in the cage after regaining consciousness and standard animal care was maintained throughout the process. Weekly booster doses, prepared as indicated in Table 1, were administered on D7, 14 and 28.
Blood was drawn from the immunized rat to obtain serum on Day 0 (pre bleeding), 7, 14, 21, 28, 35 and 42. On each occasion, approximately 450 μl of blood was drawn by tail snipping of the anaesthetized rat. The collected blood was allowed to clot for 1 h at RT and thereafter at 4 °C for 30 min after disturbing the clot. The clotted blood sample was then spun at 950×g for 10 min and serum was collected into sterile Eppendorf tubes and stored at − 20 °C until further use.
Each serum sample was de-complemented prior to use for surface immunofluorescence assay, by keeping for 30 min at 56 °C water bath. The de-complemented sera were kept stored at − 20 °C until used.
Surface immunofluorescence assay. Immunofluorescence assay was performed as described below following the method detailed by Camargo 12 , using the pre-prepared egg antigen slides mentioned above. Rat  www.nature.com/scientificreports/ anti-serum against E. vermicularis egg antigens or serum from an individual with chronic enterobiasis were used as the primary antibody, which was then detected by using commercially available fluorescein isothiocyanate (FITC) conjugated goat anti-rat IgG antibodies as the secondary antibody and the slides were examined under a fluorescence microscope. The egg antigen slides were thawed under dehydrated conditions and each well was incubated with 1:4 dilution of human and rat immune sera in PBS for 45 min at RT. Wells were washed three times with PBS, and thereafter, incubated with 1:40 dilution of the secondary antibody in PBS (goat anti-rat FITC conjugated IgG antibody) for 30 min in the dark at RT. Wells were re-washed with PBS three times in the dark, and then covered with a coverglass and examined under the fluorescence microscope.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE was
performed according to the procedure described by Laemmli 13 , and consisted two layers of gel; a resolving (separating) gel layer at the bottom and a stacking gel layer at the top. About 5 ml of 12% acrylamide was used for the separating gel and 2 ml of 5% acrylamide for the stacking gel.
Samples were prepared as follows. Ten microlitres of egg protein at a concentration of 400 μg/ml was mixed with the gel loading buffer at 1:1 ratio and incubated for 5 min at 95 °C. Up to 150 μl of the sample was loaded per well followed by 10 μl of un-stained marker. The gel was run with 1× Tris-Glycine electrophoresis buffer at 70 V for approximately 150 min until the dye front reached the bottom of the gel. A portion of gel containing the protein bands (two lanes with protein marker and a single lane with egg antigens) was removed and silver stained. Remaining portion of gel containing a broad lane of egg antigens was used for the western blot.
Silver staining was done as follows. The gel was firstly incubated in fixing solution containing ethanol, glacial acetic acid and distilled water at a ratio of 30:10:60 respectively for 1 h with gentle shaking. After 30 min, the fixing solution was removed and the gel was incubated in washing solution of 50% methanol in water. The washing solution was then discarded and the gel was incubated in distilled water (rinsing solution), twice for 10 min on each occasion. After removing the rinsing solution, a sensitizing solution of 0.02% sodium thiosulfate in water was added and incubated for 1 min. Then the solution was discarded and the gel was rinsed with distilled water twice, lasting for 1 min each time. After removal of the rinsing solution, the gel was incubated with the freshly prepared staining solution of 0.1% silver nitrate in water for 30 min. After incubation, the gel was washed with de-ionized water and again incubated in freshly prepared developing solution of 2.5% sodium carbonate and 0.02% formaldehyde in water. The reaction was stopped by washing the gel in 1% acetic acid followed by deionized water.

Molecular weight estimation of proteins in SDS-PAGE gel. Molecular weight estimation was done
by plotting the ratio of the migrated distance (in cm) of protein standard of the pre-stained molecular marker to the dye front (R f value) and the log values of molecular weights of the protein standards. BenchMark (Thermo Fisher Scientific, USA) unstained protein ladder was used as the standard molecular weight marker and the dye front of 4.7 cm from the silver staining procedure were used for calculation of R f values ( Table 2). The graph plotted using the calculated R f values and log molecular weights is shown in Fig. 2.
Molecular weights of the E. vermicularis egg protein antigens were then determined by calculating the R f values of each band and comparing them with the calibrated molecular weight markers.

Western blot analysis.
Detection of specific immunogenic proteins in the egg antigens was done using western blotting 14 . The gel containing waffle was placed accurately in the electrode assembly, then closed tightly in the transfer tank with an ice pack, and connected to a power supply. The proteins in the gel were transferred to the nitrocellulose membrane for 2½ h at 70 V.

Protein concentration of the E. vermicularis egg antigen sample. Concentration of the egg sample
was 8000 eggs/ml with an average OD 595nm of 0.0805, and the total protein concentration of the crushed egg antigen sample was estimated to be 394 µg/ml.

Surface immunofluorescence.
Surface immunofluorescence was observed only with rat sera obtained from day 14 till day 42 post-inoculation, indicating that sufficient IgG had formed only by day 14 post-inoculation ( Fig. 3A-F). The sample of human serum too gave positive results (Fig. 3G).

Molecular weights of the E. vermicularis egg antigens.
About 20 protein bands were detected with approximate molecular weights ranging from 18 to 151 kDa (Fig. 4, Supplementary File S1).
Western blot analysis. The secondary antibody dilutions from each incubated strip turned purple on addition of the substrate and the coloured bands that became visible are shown in Fig. 5. Images were edited for brightness, contrast and colour using picture format tools of MS Word to obtain better clarity (un-edited images are shown in Supplementary File S1). The distances travelled by these blotted bands on the filter papers and their estimated relative molecular weights are given in Table 3. These bands which blotted on the membrane are representative of the immunogenic egg antigens of E. vermicularis and would no doubt be useful in future studies directed towards the development of immunodiagnostics for enterobiasis.

Discussion
Microscopic examination of perianal swabs is the standard method used for diagnosis of enterobiasis 15 . However, the variable and nocturnal nature of egg deposition on the perianal skin by the gravid female E. vermicularis worm requires samples to be acquired early morning prior to washing of the perianal region on at least three consecutive days for accurate diagnosis 16,17 . In addition, the swabs need to be examined microscopically by an experienced individual for identification of pinworm eggs, which is rather time consuming. An immunodiagnostic method which is easy to perform with equal or more sensitivity and specificity to microscopy, would be most useful in the treatment and control of this infection. The current study was conducted to identify potential antigens of E. vermicularis as a preliminary step towards the development of such an immunodiagnostic tool.
Clinical manifestations of enterobiasis are generally quite suggestive of infection and routine diagnostic tools are fairly reliable 15 . However, many infections may be asymptomatic, with ongoing underlying transmission occurring at individual as well as community level. As healthcare becomes more consumer-focused, the need for convenient and effective point-of-care diagnostics is expanding worldwide. A point-of-care diagnostic tool for enterobiasis could allow for screening and initiation of treatment during a single encounter. In Sri Lanka, mass deworming of schoolchildren conducted through annual school medical inspections for over five decades has been successful in controlling soil-transmitted helminth infections (< 1%) in the country 18 . However, the prevalence of enterobiasis continues to remain over 20% amongst these schoolchildren 3 . As such, a point-of-care www.nature.com/scientificreports/ diagnostic tool would improve the ease in conduct of epidemiological surveys required to inform public health policy and for implementation of population targeted interventions. Conduct of the current study for identification of immunogenic surface antigens of E. vermicularis eggs was quite challenging due to the lack of scientific literature in this regard. Commencing from the extraction of eggs from perianal swabs, to purification, disruption and then finally the detection of immunogenic antigens required experimentation with many processes and documentation of them in this paper would no doubt be helpful for future research activities in this area.
Scratching off eggs manually from the adhesive cellophane swab was not successful, while absolute alcohol for egg removal was inefficient and caused whitish discolouration of eggs. Absolute acetone (propan-2-one) which is the simplest and smallest ketone, was the best at harvesting these eggs. Acetone exhibits only slight toxicity and was therefore more suitable than other solvents for harvesting pinworm eggs from these adhesive www.nature.com/scientificreports/ cellophane swabs. Furthermore, purification by serial washing with acetone was also useful in removing any remaining debris and glue flakes from the isolated eggs. Disruption of the hard and thick egg shell required experimenting with various egg disruptive methods prior to achieving success. Mini spin centrifugation at high centrifugal force for short time periods only resulted in clumping of eggs, while just vortexing or sonicating of eggs was not effective either. Similarly, Triton X-100, which is a widely used detergent to lyse cells for extraction of proteins and organelles and to permeabilize living cell membranes 19 nor SDS, which is an anionic detergent with a negative charge that usually can denature native   www.nature.com/scientificreports/ proteins by disturbing of non-covalent forces, could disrupt the egg shells successfully. With SDS, it was noted that larval stages did emerge partly from the eggs, but the toxic nature of SDS was thought likely harmful for the rat and to interfere with development of immune sera. Continuous freeze thaw cycles known to damage cells 20 could not significantly disrupt these pinworm eggs. Finally, the eggs were effectively crushed only with the use of glass beads, together with sonication prior to vortexing helpful in saving time. Surface immunofluorescence of intact pinworm eggs was clearly observed after reacting with immune serum developed in the Wistar rat as described by Wakayama et al. 21 . Reactivity was observed with sera obtained at day 14 post-inoculation and thereafter, indicative of the presence of immunogenic antigen expression on the surface of pinworm eggs, despite being covered in a hard shell. The positive fluorescence that emanated from human serum of the donor with chronic enterobiasis confirmed that these surface antigens are capable of producing an immune response upon exposure to the host immune system. Surprisingly, this same sample of human serum did not react with the protein bands in the immunoblotting probably due to its greater dilution in this assay (1:25) when compared to that used in the immunofluorescence assay (1:4).
The life cycle of E. vermicularis in humans is usually confined to the gut, with onset of infection resulting from ingestion of infective eggs that are deposited on the perianal skin by the gravid female worm 4 . In chronic pinworm infection, repeated exposure to intact eggs, larval stages and adult worms would have occurred in the gut. For the development of immune sera in the rat, crushed eggs were injected subcutaneously, which was successful in yielding an immune response against both intact (immunofluorescence assay) as well as crushed (immunoblotting assay) egg antigens. It thus appears likely that these immunogenic antigens are shared between the different stages of E. vermicularis. Furthermore, our results confirm the presence of serum antibodies in persons with chronic enterobiasis.
A total of 20 protein bands of E. vermicularis eggs were identified and their approximate molecular weights determined by using SDS-PAGE assay, while their immunogenicity was assessed through immunoblotting with rat antiserum. The most prominent and immunogenic protein bands identified in this study comprised of 8 proteins with molecular weights that ranged between 21 and 151 kDa. The development pattern of IgM and IgG antibodies in rat serum were identified through the immunoblot (Table 3) and conformed to established timelines. IgM antibodies are known to develop within the first two weeks, with peak levels usually on day 7 11 . In our study, IgM against egg antigens were observed on day 7, and remained until day 14-21 in some; while IgG was mainly observed from day 21 onwards and continued to be present until the last day of observation (day 42). If these results can be extrapolated to humans, the presence of specific IgM may be exploited to detect current infection, while IgG would be indicative of exposure to pinworm infection. Autoinfection is common with E. vermicularis, and infection is generally known to last for long periods once established due to continued re-infection 22 , thus allowing for the detection of specific IgG. However, the demonstration of more than twofold rise in titres of specific IgG may be required to differentiate between current and past infection.
The possibility of detecting these immunogenic antigens in blood or other human tissues needs further study. In-depth analysis on the presence and detection of these immunogenic antigens in various body tissues, the duration of their availability, as well as specificity (with no cross reactivity to other helminth antigens) may provide an opportunity for the design and development of novel, rapid, point-of-care diagnostics for E. vermicularis infection. Furthermore, recent studies have discovered the immune-modulation properties of tissueinvasive helminths and their excretory-secretory molecules, which are now being exploited for use as immune therapeutics [23][24][25][26] . Several studies have suggested an immune-protective role of E. vermicularis infection against allergies [27][28][29] . As such, understanding the effects yielded by these E. vermicularis antigens on the host immune system (including gut mucosal immunity) may create opportunities for the design of novel therapeutics and vaccines. www.nature.com/scientificreports/ In conclusion, the methods used in this study were effective in obtaining E. vermicularis egg antigens which were immunogenic. Furthermore, surface antigens of intact eggs reacted with antibodies developed against crushed egg antigens. These findings may pave the way for the development of effective immunodiagnostics.

Data availability
All data generated or analysed during this study are included in this published article [and its Supplementary Information files].