Jpn. J. Infect. Dis., 71, 116–121, 2018
Original Article Venom and Antivenom of the Redback Spider (Latrodectus hasseltii) in Japan. Part I. Venom Extraction, Preparation, and Laboratory Testing Takayuki Matsumura1, Reona Mashiko2, Tomomi Sato2, Kentaro Itokawa2, Yoshihide Maekawa2, Kohei Ogawa2, Haruhiko Isawa2, Akihiko Yamamoto3, Shigemi Mori4, Akira Horita4, Akihiro Ginnaga4, Yoshinobu Miyatsu4, Motohide Takahashi5, Hisashi Taki6, Toru Hifumi7, Kyoko Sawabe2†, and Manabu Ato1*† 1Department of Immunology, National Institute of Infectious Diseases; 2Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo 162-8640; 3Division of Biosafety Control and Research, National Institute of Infectious Diseases, Tokyo 208-0011; 4The Chemo-Sero-Therapeutic Research Institute (Kaketsuken), Kumamoto 860-8568; 5Pharmaceuticals and Medical Devices Agency, Tokyo 100-0013; 6Ministry of Health, Labour and Welfare, Tokyo 100-8916; and 7Kagawa University Hospital Emergency Medical Center, Kagawa 761-0793, Japan
SUMMARY: The redback spider (Latrodectus hasseltii Thorell) reportedly invaded Japan in September 1995. To date, 84 redback spider bite cases have been reported; 7 of these cases employed the antivenom. Antivenom has been imported from Australia in the past, but because of restrictions on exportation it was evident that nearly all of the antivenom present in Japan would expire during 2014. In 2014, a plan was proposed to experimentally manufacture and stockpile a horse antiserum for ourselves, using redback spiders indigenous to Japan. A total of 11,403 female spiders were captured alive: 1,217 from the vicinity of Nishinomiya City, Hyogo prefecture, and 10,186 from Osaka prefecture. Of these, 10,007 females were dissected, and the venom was extracted from the venom glands of individuals and subjected to crude purification to yield 4 lots, of which the majority was α-latrotoxin. Among them, a large amount of single lots with an estimated protein content of 236 mg is subsequently scheduled to be used for immunizing horses. We also determined lethal toxicity of the venom (LD50: 9.17 μg per mouse), and established the assay for the determination of an anti-lethal titer of antivenom in mice.
in recent years, importation from Australia has been INTRODUCTION limited. As a result, it became clear that nearly all of the In September 1995 the redback spider (Latrodectus antivenom present in Japan would expire during 2014. hasseltii Thorell) was first discovered in Takaishi City, A plan for experimental production and stockpiling Osaka prefecture (1, 2), in Yokkaichi City, Mie prefecture of a horse antiserum in Japan was implemented using the following November, and in Miyakojima City, redback spiders indigenous to Japan. We captured Okinawa prefecture that December (3). The distribution redback spiders, extracted their venom glands, and of redback spiders in Japan has since expanded. It has crudely purified the venom. We then performed been shown to be established in a total of 43 prefectures characterization analysis, determined the lethal toxicity in Japan until 2016 (4). The possibility that the of the venom (LD50), and established the assay for the distribution of this spider will continue expanding and determination of anti-lethal titer of antivenom in mice. that the population will continue increasing are causes for concern. MATERIALS AND METHODS Since 1996, 84 redback spider bite cases have been reported (14 of these cases were reported in 2009), and Capture of redback spiders: A total of 11,403 7 of these cases employed antivenom (5 and T. Hifumi, female redback spiders were captured alive: 1,217 in unpublished data). Native to Australia, the redback the vicinity of Nishinomiya City, Hyogo prefecture, and spider is known as a venomous species; there are 3,000 10,186 in Osaka prefecture (Fig. 1A and 1B). From June to 5,000 bite cases every year (6), and an antivenom is through December 2014, female spiders were captured also used for the treatment. The antivenom produced in alive in several sites close to Nishinomiya City and in Australia has been imported for use in Japan. However, 480 sites in Osaka prefecture. Every effort was made to capture individuals alive by hand using tweezers or with Received June 29,2017. Accepted November 6, 2017. gloves. Pesticides were not used during capture, and J-STAGE Advance Publication February 28, 2018. specimens were killed by freezing at –20°C. The redback DOI: 10.7883/yoken.JJID.2017.291 spiders stored in the freezer were transported frozen in *Corresponding author: Mailing address: Department of various batches to the National Institute of Infectious Immunology, National Institute of Infectious Diseases, Diseases (NIID), Tokyo, Japan. Dry ice was used during 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel: transport to prevent specimens from thawing. +81-3-5285-1111, Fax: +81-3-5285-1147, E-mail: ato@ Removal of the redback spider venom glands and nih.go.jp crude purification of the venom: Because the venom †These authors contributed equally to this work. gland is covered with a thick layer of muscle, the fangs
116 Venom of Redback Spider in Japan
B A Hyogo Tokyo
Osaka
3,403
2,223
Takaishi City 1,217 N 20 km 0 or not inves gated 1 - 100 101 - 1,000 1,001 < N 20 km
Fig. 1. Map of Hyogo (A) and Osaka (B) prefectures showing locations of the collection sites of redback spiders and their density for the entire survey period. Over 1,000 of redback spiders were captured from the black area, 101–1,000 spiders from the gray area, 1–100 spiders from the silver area and no spider was captured or not investigated at the white area. were gently seized and extracted from each spider determined using a Pierce Bicinchoninic acid (BCA) using sterilized forceps on a hole slide glass under a protein assay kit (Thermo Fisher Scientific, Rockford, stereomicroscope according to the previously described IL, USA). The neurotoxin α-Latrotoxin from Latrodectus method (7). The venom glands were individually tredecimguttatus was used as a positive control and was removed from the body and transferred to a glass slide obtained from Enzo Life Sciences (Farmingdale, NY, with wells filled with cold physiological saline (Otsuka USA). Equal amounts of protein were suspended in Pharmaceutical Co., Ltd., Tokyo, Japan). One-hundred an SDS sample buffer. The lysates (0.5 µg of positive pairs of venom glands were collected in a tube (2 ml control or 10 µg protein per lane) were boiled for 5 min protein adsorbing controlled cryopreservation tubes, then separated by SDS-PAGE and either visualized with Assist Co., Ltd., Tokyo, Japan) containing 1 ml of an Imperial protein stain kit (Thermo Fisher Scientific,) physiological saline, and stored in a freezer at –80°C or transferred to polyvinylidene fluoride membranes until use. (Millipore, Bedford, MA, USA). The membranes were All venom glands were removed, and after collection blocked with Tris-buffered saline containing 5% bovine into approximately 100 tubes, 3 zirconia beads 4 mm in serum albumin and 0.1% Tween 20 and incubated with diameter were put into each tube, and were homogenized anti-α-latrotoxin polyclonal Ab, which was developed using a high-speed mechanical homogenizer (Tissue- in rabbits using highly purified α-latrotoxin from Lyser II, QIAGEN N.V., Venlo, The Netherlands) for Latrodectus tredecimguttatus (8) (Sigma-Aldrich 30 s at 25.0 cycles per second. The tubes containing the Corp., St. Louis, MO, USA). The membranes were homogenized venom glands were centrifuged at 10,000 then incubated with anti-rabbit IgG Ab linked to HRP rpm (KUBOTA 3740, KUBOTA Co., Tokyo, Japan) (GE Healthcare Life Sciences, Buckinghamshire, UK). for 3 min, and the supernatant was then recovered and Immunoreactive proteins were visualized with an ECL poured into a 50 ml tube (Proteosave® SS centrifuge detection system (Thermo Fisher Scientific,) and were tube, Sumitomo Bakelite Co., Ltd., Tokyo, Japan). One analyzed by a chemiluminescence image analyzer, milliliter of cold physiological saline was added to each Image Quant LAS-4000 (GE Healthcare Life Sciences,). residue, and the mixture was again homogenized for Mice: All animal experiments were performed 30 s at 25.0 cycles per second and centrifuged for 15 according to institutional guidelines and with the min at 14,000 rpm (KUBOTA 3740). The supernatant approval of the NIID Animal Care and Use Committee from each sample was recovered as described above (Permit Numbers: 114097 and 115035). Female outbred and mixed in a 50 ml tube (mentioned above). The tube ddY mice (4-week-old) were purchased from Japan SLC containing the recovered venom was placed on ice to Inc.,(Shizuoka, Japan). Animals were maintained under minimize the activity of protease. The venom solution specific pathogen-free conditions. was stored at –80°C to avoid unnecessary freeze-thaw Assay for the determination of lethal toxicity in cycles. mouse (LD50): The assay was performed on 5 groups Quantification of protein, protein gel staining, (5 mice per group) for diluted solutions. The venom and immunoblotting: Quantification of protein was was diluted with 0.2% gelatin-phosphate buffered saline
117 (GPBS) in final concentrations of 144 (group 1), 120 September 2,595; October 2,280; November 2,144, and (group 2), 100 (group 3), 83 (group 4), and 69 µg/ December 1,283. Redback spiders are distributed widely ml (group 5). Aliquots of 0.1 ml of each dilution were throughout Osaka prefecture; 3,403 and 2,223 spiders injected into each mouse by intravenous route via the were captured in Toyonaka and Daito cities, respectively. tail. Subsequently, the number of deaths per group was A total of 11,403 redback spiders were captured from recorded for 10 d. LD50 was determined using the Probit both areas. method. Removal of redback spider venom glands and Assay for the determination of anti-lethal titer in crude purification of the venom: Of the 11,403 mouse: The assay was performed on 5 groups (3 mice captured redback spiders 10,007 females were dissected per group) for diluted solutions. Control antivenom under a stereomicroscope, and the venom glands (Red Back Spider Anti-venom; CSL, Parkville, Victoria, were extracted. Several tubes stored in a freezer at Australia) was diluted with GPBS in the concentrations –80°C were taken at random from the frozen venom of 40 (group 1), 32 (group 2), 25.6 (group 3), 20.48 glands and divided into approximately 100 tubes, and (group 4), and 16.43 U/ml (group 5). The venom before forming a large amount of single lot containing (400 µg/ml) and each diluted antivenom was mixed in small amounts of 3 lots were tentatively extracted and equal amounts for one h at room temperature. Aliquots subjected to crude purification (Lot. 140919, 140930, of 0.2 ml of mixed solutions were injected into each and 141021). A protein quantification assay determined mouse by intravenous route via the tail. Subsequently, that protein concentrations of Lot. 140919, 140930, and the number of deaths per group was recorded for 10 d. 141021 were 1.57, 1.95, and 2.52 mg/ml, respectively. The quantity of protein in the crude purified venom, RESULTS when finally made into a large amount of single lot, was approximately 236 mg (Lot. 150115). The total quantity Capture of redback spiders: In Nishinomiya city in of 170 ml of the crude venom preparation (1.39 mg/ml) Hyogo prefecture female redback spiders were captured was sent frozen to the Chemo-Sero-Therapeutic from June to December 2014 (Fig. 1A). From June to Research Institute. It is scheduled to be used as an mid-July, captures were affected by typhoons and heavy immunization for horses. We then performed SDS- rains; in reality, captures were only possible from late PAGE and immunoblotting using an anti-α-latrotoxin July. A total of 1,217 female spiders were captured polyclonal antibody which developed in rabbits using from the vicinity of Nishinomiya City. While in Osaka highly purified α-latrotoxin from L. tredecimguttatus prefecture a total of 410 members of the Osaka Pest (black widow) (8). The α-latrotoxin isolated from L. Control Association were called out, and carried out the tredecimguttatus was used as a positive control (Fig. spider capture from June to December 2014, at a total of 2A). The results of immunoblotting and protein gel 480 sites (Fig. 1B). By late December, a total of 10,186 staining indicated that α-latrotoxin (molecular weight: female spiders had been captured. The numbers captured ~130 kDa) (9) was the predominant protein in the in each month were as follows. July 528; August 1,356; A A A B B B venom, stable for 2 dC atC roomC temperature, for 4–12 ProtPerinoteiPnrotein A B ImmunoCImmuno- Immuno- gel-gel gel ImmunobImmunobImmunoblo lnog nglo ng ImmunobImmunobImmunoblo lnog nglo nPgrotPerinot geeiPnlr sogtteainingel isnt aininggel staining blo blngo ngblostainingngstainingstaining (kDa()kDa) (kDa) (kDa()kDa) (kDa) 250 250 250 250 250 250 (kDa()kDa) (kDa) 250 250 250 150 150 150 150 150 150 150 150 150 100 100 100 100 100 100 100 100 100 75 75 75 75 75 75 75 75 75 50 50 50 50 50 50 50 50 50 37 37 37 37 37 37 37 37 37
25 25 25 25 25 25 25 25 25 20 20 20 20 20 20 20 20 20 15 15 15 15 15 15 15 15 15 10 10 10 10 10 10 10 10 10 5 5 5 5 5 5 2 2 2 5 5 5 2 2 2 2 2 2 r r r r r r k k k k k k y y y k k k y y k k y k k k k y y y n n n a a a a a a i i i a a a a a a a a a e e e e e e e e e e e e e e e x x x e e e e e e d d d d d d d d d e e e e e e e e e e e e e e e y y y y y y o o o ------t t t w w w w w w w w w w w w w w w 2 2 2 2 2 2 2 2 2 ------
2 2 2 2 2 2 o o o
r r r 2 2 2 T T T 4 4 4 T T 4 4 2 2 T 4 2 T T 4 4 T 4 140919 140919 140930 140930 141021 140919 141021 140930 141021 141021 141021 141021 150115 150115 141021 141021 150115 141021 150115 150115 150115 150115 150115 150115 150115 150115 150115
t t t C C C C C C ° ° ° ° ° ° 1 1 1 1 1 1 R R R R R R R R R a a a
C C C C C C C C C
° ° ° ° ° ° ° ° ° L L L C C C C C C ° ° ° ° ° ° - - - 4 4 4 4 4 4 4 4 4
4 4 4 4 4 4 α α α –80 –80 –80 –80 –80 –80
141021 141021 141021 141021 141021 141021 141021 141021 141021 140919 140919 140919 141021 141021 141021 141021 141021 141021 141021 141021 141021 141021 141021 141021 150115 150115 150115 150115 150115 150115
Fig. 2. Immunoblotting and protein gel staining of venom of redback spider. (A) α-Latrotoxin from black widow spider (0.5 µg, positive control), Lot. 140919 (control and 4°C at 4 weeks), 140930, 141021 (control and room temperature at 2 days), and 150115 of venom (10 µg) of redback spider were analyzed by immunoblotting. (B, C) Stability test of venom of redback spider. (B) Lot. 141021 (control, room temperature at 2 days, 4°C at 4 weeks, and 4°C at 12 weeks) and Lot. 150115 of venom (10 µg) of redback spider were analyzed by immunoblotting and protein gel staining. (C) Lot. 150115 (control and –80°C at 2 years) of venom (10 µg) of redback spider were analyzed by immunoblotting and protein gel staining.
118 Venom of Redback Spider in Japan
Table 1. Determination of lethal toxicity of venom in mice Number of deaths Assay Venom (μg/mouse) day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 9 day 10 total I 14.4 1 3 1 5/5 12 0 3 2 5/5 10 0 1 1 1 1 1 5/5 8.3 0 0 0 0 0 0 0 0 0 0 0/5 6.9 0 0 0 0 0 0 0 0 0 0 0/5 II 14.4 0 3 2 5/5 12 0 3 2 5/5 10 0 2 1 0 0 0 0 0 0 0 3/5 8.3 0 0 0 0 0 0 0 0 0 0 0/5 6.9 0 0 0 0 0 0 0 0 0 0 0/5 III 14.4 0 3 2 5/5 12 0 1 2 1 0 1 5/5 10 0 1 2 0 1 0 0 0 0 0 4/5 8.3 0 1 1 1 0 0 0 0 0 0 3/5 6.9 0 0 0 0 0 0 0 0 0 0 0/5
LD50 (average of 3 assays): 9.16899 μg/mouse (I: 9.22588 μg/mouse, II: 9.80604 μg/mouse, III: 8.47505 μg/mouse).
Table 2. Assay for the determination of anti-lethal titer in mouse Number of deaths Assay Antivenom Venom (U/mouse) (μg/mouse) day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 9 day 10 total I 4 40 0 0 0 0 0 0 0 0 0 0 0/3 3.2 40 0 0 0 0 0 0 0 0 0 0 0/3 2.56 40 0 0 1 2 3/3 2.048 40 0 0 1 2 3/3 1.643 40 1 2 3/3 II 4 40 0 0 0 0 0 0 0 0 0 0 0/3 3.2 40 0 0 0 0 0 0 0 0 0 0 0/3 2.56 40 0 1 1 0 1 3/3 2.048 40 0 0 1 2 3/3 1.643 40 0 0 3 3/3 weeks at 4°C or for –80°C at 2 years, and there was little for 10 d after intravenous injection. All mice that difference among the 4 lots (Fig. 2A, 2B, and 2C). received the injected mixture of 40 µg of venom and 3.2 Toxicity of venom in mice: Five ddY mice (female) U or 4 U of antivenom fully recovered in 2 independent weighing 16–18 g were used. They were observed for experiments (Table 2). These results suggest that CSL 10 days after intravenous injection of the venom from antivenom in Australia could use as control antivenom the redback spider (6.9, 8.3, 10, 12, or 14.4 µg per for the determination of anti-lethal titer of antivenom mouse). All mice died 3–6 d after high-dose injections candidates using the venom of redback spider in Japan. (12 and 14.4 µg per mouse). The mice were crouching with closed eyes and benumbed legs before they died. DISCUSSION However, all mice that received low doses (6.9 µg per mouse) survived and did not exhibit clinical symptoms. Of the 84 cases of redback spider bite in Japan, to date The LD50 value was 9.17 µg per mouse, calculated as the 6 cases have employed an antivenom, which is hardly mean value obtained from 3 independent experiments a large number. In general, the skin reaction at the site (Table 1). bitten by Latrodectus spp. is not especially severe, and Neutralization of venom by antivenom: The venom in some cases no erythema is produced with the only of the redback spider in Japan was neutralized by CSL indication of a bite is the presence of one or 2 puncture antivenom (Parkville, Victoria, Australia) in Australia marks. However, signs and symptoms such as pain, when they were mixed for one h in vitro. In CSL nausea, vomiting, abnormal sweating, fatigue, sensory antivenom, 500 U of antivenom has been standardized abnormalities, and fever might be observed in addition to neutralize 5 mg of venom according to the attached to irritation of the bite site. There were several reports of data sheet. This is because it is defined when the deaths before the commercial availability of antivenom redback spider antitoxin of 1 U neutralizes 0.01 mg of in Australia. For this reason, the administration of toxins (10). Three mice were injected with the mixture antivenom to bite victims is being studied in Japan as containing 40 µg of the venom (equivalent to 4 × LD50 well as being prescribed in practice. Since it is predicted per mouse) and several doses of the antivenom (1.643, that the serum will also be utilized in the future, 2.048, 2.56, 3.2, or 4 U per mouse). They were observed planning the experimental production and stockpiling of
119 redback spider antivenom in Japan is a valid choice. indicate that α-latrotoxin, with a molecular weight of Since its first sighting in September 1995 in the Osaka approximately 130 kDa, had been degraded; therefore, it Bay area, Takaishi City (Fig.1B) (1, 2), the redback is assumed that a certain degree toxicity was maintained. spider has spread throughout Japan with the exception When forming a single lot, it is important that of Aomori, Akita, Nagano, and Toyama prefectures, repeated freezing and thawing does not occur, and that as of the end of 2016 (4). Based on the geographic tasks are carried out at low temperature; therefore, the information systems (GIS) analysis using data from task was completed in a single day, with transport on the 1995 to 2001, the distribution of this spider clearly following day, still in the cold stored condition, to the continues to expand in some coastal areas and the Chemo-Sero-Therapeutic Research Institute. inner areas of Osaka prefecture, but not in the northern We next determined lethal toxicity of the venom (LD50: area (11). During the initial 13 years, the distribution 9.17 µg per mouse). The toxicity of α-latrotoxin is expanded and increasingly more spiders were found varied among the reports; Australian group has reported in the northern and inner areas of Osaka prefecture. In that LD50 of the redback spider venom to mice was 0.43 the neighboring Hyogo prefecture, the first discovery mg/kg in 1964 (15). In Japan, Osaka Prefecture Institute of the redback spider was September 1997, and the of Public Health had reported the LD50 of venoms from spider has since been found in 13 cities and one town spiders which were captured around Osaka area in 1996 (4). Over 1,000 spiders were captured in Nishinomiya was evaluated three times during 1996–1998, which was City in 2014. There have been several reports recently varied from 0.22 to 0.91 mg/kg (16). In 1996 (LD50 was that large numbers of the redback spider have been 0.22 mg/ml), they described that LD50 of lyophilized found in service areas of the Chugoku Highway in standard venom provided by CSL, as an internal Hiroshima prefecture. Such observations suggest that the control, is accounted for 0.477 head of the spiders, development of the transportation network has greatly which could be estimated as ~0.5 mg/kg (17). In this contributed to the expansion of the distribution range study, we determined LD50 of redback spider venom of this spider. It is clear that as the spider population that we collected to 4 weeks old female ddY mice as increases, the number of spider bites also increases. ~0.5 mg/kg. These suggest that the toxicity of redback A series of works such as capturing and transportation spider venom in Japan has not different among that from of redback spiders, and crude purification of the venom spiders in Australia. was an unprecedented attempt in Japan. For capturing α-Latrotoxin is a potent neurotoxin that triggers the redback spiders, a method with live capture without synaptic vesicle exocytosis from presynaptic nerve using a pesticide, and killing by freezing, was proposed. terminals (18). It is a high-molecular mass neurotoxin In general, pesticides are most often used in order to and the predominant protein in the venom of the capture the spiders, with the objective of killing them. redback spider. When mice were injected with high However, for the purpose of extracting protein venom doses of the venom, they were crouching with closed it was considered necessary to make every effort eyes and benumbed legs before dying, suggesting to eliminate chemical contamination. Since several that α-latrotoxin presynaptically affects the nervous hiding places for redback spiders are out of reach of system. Using the venom of the redback spider and tweezers or fingers, capture by this method was initially CSL antivenom, we finally established an assay for difficult. Moreover, since it was foreseen that when the determination of anti-lethal titer of antivenom put into an insect bottle in large numbers redback candidates. Our assay was performed to determine anti- spiders they would eat each other, or venom would be lethal titer of antivenom candidates at least 10 times, and released, etc., and hinder subsequent tasks, care was we were able to confirm that the sensitivity, stability, taken with temporary storage until reaching the nearest and reproducibility of that are sufficient (19). administrative office. When transporting redback spiders to NIID, the utmost care was necessary to prevent the Acknowledgements This research was supported by the specimens from thawing during transport. Research Program on Emerging and Re-emerging Infectious In this study, crude venom of redback spiders was Diseases from Japan Agency for Medical Research and prepared with reference to the previous method (12). Development, AMED. For the capture of redback spiders in Osaka prefecture, a total of 410 members of the Osaka Pest Control However, since details were not described in the text, Association were called out. The authors sincerely thank their we then established a new method for preparing crude contributions. We also thank Mr. Shizuo Yonesho, Mr. Tsunehiro venom of redback spiders used for immunization against Taira and Mr. Kunihiro Okada of Environmental Sanitation horses. It is known that α-latrotoxin, the principal Division Nishinomiya City Office for the capture of the spiders constituent of redback spider venom, is deactivated by in Nishinomiya City. Mr. Kyohei Yamamoto of Meiji University, proteases. Accordingly, addition of Tris-HCl buffer or an Drs. Mutsuo Kobayashi, Yoshio Tsuda and Hiroko Ejiri of the enzyme inhibitor (13, 14) in tasks subsequent to removal Department of Medical Entomology, NIID, for their considerable of the venom glands was initially studied; however, collaboration in removal of the redback spider venom glands. considering the fact that in the end horses are to be immunized, it was decided to extract with physiological Conflict of interest S.M., A.H., A.G., and Y.M. are employees of The Chemo-Sero-Therapeutic Research Institute. The authors saline. Therefore, tasks at room temperature were otherwise have no financial conflicts of interest. done in the shortest possible time, and care was taken to place the physiological saline and storage tubes used in ice throughout the duration of the extraction. REFERENCES Fortunately, the quantity of protein in the venom in 1. Kanazawa I, Nishikawa Y, Tomizawa O. Introduction of the redback randomly selected storage tubes was calculated to be spiders to Japan. 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