Concurrent Ascaris suum and Oesophagostomum dentatum infections in pigs

Veterinary Parasitology 82 (1999) 221±234

Concurrent Ascaris suum and Oesophagostomum dentatum infections in pigs A.B. Helwigh*, C.M. Christensen, A. Roepstorff, P. Nansen Danish Centre for Experimental Parasitology, Royal Veterinary and Agricultural University, Ridebanevej 3, DK-1870 Frederiksberg C, Copenhagen, Denmark Received 22 September 1998; accepted 20 January 1999

Abstract The aim of this study was to examine interactions between Ascaris suum and Oesophagostomum dentatum infections in pigs with regard to population dynamics of the worms such as recovery, location and length; and host reactions such as weight gain, pathological changes in the liver and immune response. Seventy-two helminth-naõÈve pigs were allocated into four groups. Group A was inoculated twice weekly with 10 000 O. dentatum larvae for 8 weeks and subsequently challengeinfected with 1000 A. suum eggs, while Group B was infected with only 1000 A. suum eggs; Group C was inoculated twice weekly with 500 A. suum eggs for 8 weeks and subsequently challengeinfected with 5000 O. dentatum larvae, whereas Group D was given only 5000 O. dentatum larvae. All trickle infections continued until slaughter. Twelve pigs from Group A and B were slaughtered 10 days post challenge infection (p.c.i.) and the remaining 12 pigs from the each of the four groups were slaughtered 28 days p.c.i.. No clinical signs of parasitism were observed. The total worm burdens and the distributions of the challenge infection species were not influenced by previous primary trickle-infections with the heterologous species. Until day 10 p.c.i. the ELISA response between A. suum antigen and sera from the O. dentatum trickle infected pigs (Group A) pigs were significantly higher compared to the uninfected Group B. This was correlated with a significantly higher number of white spots on the liver surface both on Day 10 and 28 p.c.i. in Group A compared to Group B. The mean length of the adult O. dentatum worms was significantly reduced in the A. suum trickle infected group compared to the control group. These results indicate low level of interaction between the two parasite species investigated. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Ascaris suum; Oesophagostomum dentatum; Pig-nematoda; Concurrent infections; Interaction

* Corresponding author. Tel.: +45-35-282-789; fax: +45-35-282-774; e-mail: [email protected] 0304-4017/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 9 ) 0 0 0 0 7 - 2

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1. Introduction Most experimental studies on parasite infections in definitive hosts are based on infections with a single species. This is done despite the fact that in nature hosts with more than one parasite seem to be the rule rather than the exception (Buck et al., 1978; Gbakima, 1994; Ferreira et al., 1994). Concurrent infections may result in antagonistic or synergistic interactions as reviewed by Holmes (1972) and Christensen et al. (1987). Interactions may be observed between closely related species (Dash, 1981; Christensen et al., 1997; Dobson and Barnes, 1995), or between species living in the same organs of the host (Holmes, 1961; Monrad et al., 1981) or even between distantly related species living in different organs of the host (Campbell et al., 1977; Kloosterman and Frankena, 1988). Most studies on heterologous interactions between helminths of livestock have focussed on cattle and sheep (Borgsteede, 1981; Dash, 1981; Reinecke et al., 1982; Satrija and Nansen, 1993; Dobson and Barnes, 1995), while little attention has been paid to possible interactions between helminths coexisting in the pig. In Danish swine herds, Ascaris suum and Oesophagostomum spp. are the most common helminths found (Roepstorff and Jorsal, 1989; Roepstorff, 1991). As reviewed by Roepstorff and Nansen (1994) parasite occurrence is strongly dependent on the type of management system used, with more parasite species and higher infection intensities being found in outdoor reared animals as compared to intensive indoor systems. Roepstorff et al. (1992) found that on almost all organic farms pig populations were infected with both A. suum and Oesophagostomum spp. Both species have a cosmopolitan occurrence, and under extensive farm conditions high infection levels and concurrent infections may exist (Roberts, 1940; Jacobs and Dunn, 1969; Ajayi et al., 1988). Although the adult stages of the two species are located in different parts of the intestinal tract, a recent experimental study (Murrell et al., 1997) has shown that newly hatched larvae of A. suum penetrate the wall of the large intestine at the predilection site of O. dentatum, rather than penetrating the wall of the small intestine, as previously thought. High doses of O. dentatum cause severe changes in the wall of the large intestine in pigs (Christensen et al., 1995) which may inhibit the migration of a challenge infection with A. suum. The penetration of high numbers of A. suum larvae through the large intestine may cause changes in the wall and thereby, inhibit the migration of O. dentatum into the wall. Therefore, the objective of the present study was to provide knowledge about interactions between A. suum and O. dentatum by investigating the population dynamics of a challenge infection with either parasite species after a period of trickle infections with the heterologous parasite. Parameters such as worm recovery, worm length and location in the host together with host reactions such as pathological changes of the liver and immune response were investigated. 2. Materials and methods 2.1. Experimental animals, housing and feeding Seventy-two helminth-naõÈve Danish Landrace/Yorkshire/Duroc cross-breed pigs (36 castrated males and 36 females from 12 litters), were allocated into four groups according

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to litter, sex and weight. The pigs, which had been shown to be helminth free by repeated faecal examinations, were raised at a specific-pathogen-free research farm of the Federation of Danish Slaughterhouses, jointly administered by the Danish Pig Breeders Organization and the Royal Veterinary and Agricultural University. At the start of the experiment the pigs were 6±9 weeks old and had an average body weight of 19.4  3.3 kg. The four groups were housed in the same building and all the pigs were penned individually. Precautions were taken to prevent cross-contamination between the pigs by having the caretakers use separate tools and boots for each group in the daily routine. The pigs had free access to water and were fed twice daily with ground barley and protein supplement. This diet has been shown to provide favourable conditions for the establishment and survival of helminths in pigs (Petkevicius et al., 1995). 2.2. Parasite isolates The strain of A. suum (designated CEP) was isolated in 1993 (Roepstorff and Murrell, 1997) and has since been maintained by repeated passages in helminth-naõÈve pigs. Eggs were isolated from fresh faeces and embryonated in 0.1 N H2SO4 in the dark at room temperature for at least 3 months. This embryonation method is known to result in highly infective eggs (Oksanen et al., 1990). The strain of O. dentatum (designated EH) was originally isolated in 1983 (Roepstorff et al., 1987) and has since been maintained by repeated passages in helminth-naõÈve pigs. Third stage larvae were obtained from faecal cultures on vermiculite, and subsequently stored at 108C for at least 3 weeks before inoculation, as described by Bjùrn et al. (1989). 2.3. Experimental design The four experimental groups were treated as described in Table 1. Group A was inoculated twice weekly with 10 000 O. dentatum larvae and challenge infected with 1000 A. suum eggs after 8 weeks, while the control Group B was given only 1000 A. suum eggs at challenge; Group C was inoculated twice weekly with 500 A. suum eggs and challenge infected with 5000 O. dentatum larvae after 8 weeks, while the control Group D was given only the challenge dose of 5000 O. dentatum larvae. Trickle infections were mixed in the feed and challenge infections and control-challenge infections were given by Table 1 Experimental design Group

Number of pigs

Trickle infection

Challenge infection

Slaughter (days p.c.i.)

A B C D

24 24 12 12

10 000 O. dentatum

1000 1000 5000 5000

10, 28 10, 28 28 28

500 A. suum

A. suum A. suum O. dentatum O. dentatum

Infection protocol and dosage of O. dentatum and A. suum to four study groups. Trickle infections were mixed in the fodder twice weekly until slaughter Challenge infections were given by stomach tube 8 weeks after the first trickle infection. p.c.i. ± post challenge infection.

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stomach tube. All primary trickle infections continued until slaughter. Twelve pigs from Group A and B, respectively, were slaughtered 10 days post challenge infection (p.c.i.) and the remaining 48 pigs, 12 from each group were slaughtered 28 days p.c.i.. The pigs were weighed at the start of the experiment, at Week 8 and 10 and/or at slaughter. Faecal egg counts were carried out every fortnight during the first 8 weeks of the experiment to assess the establishment of patent infections in the pigs and to certify that no crosscontamination occurred between the groups. After challenge infection faecal egg counts were carried out every 2±4 days. Blood samples were taken at the same time intervals for measurements of antibody response. 2.4. Parasitological and immunological techniques Faecal egg counts were carried out using the concentration McMaster technique described by Roepstorff and Nansen (1998) with a lower detection limit of 20 eggs per gram faeces (EPG). At slaughter the animals were stunned by using a captive bolt pistol and then exsanguinated. Pigs in all the groups, except Group D, were examined macroscopically for white spots on the surface of the liver. In all the groups, except Group D, the liver and lungs were cut into approximately 5 mm pieces using a kitchen blender. Migrating larvae were isolated from a 25% subsample of the liver tissue using the agar-gel method described by Slotved et al. (1995) and from a 25% subsample of the lung tissue using a macro Baermann technique described by Eriksen et al. (1992). The small intestine was divided into four sections of equal length in the A. suum infected pigs but was processed intact for Group D. From Group C large adult A. suum were collected manually from the small intestine. The contents ‡ washing of the samples from the small intestine were subsequently embedded in agar to isolate small ascarids with the agar-gel method as described by Slotved et al. (1997). The large intestines from pigs in Group A, C and D were divided into 5 sections: caecum, 0±20% colon, 21±40% colon, 41±60% colon and 61±100% colon. The contents of each section were thoroughly mixed and washed with 0.9% saline solution (378C), and a 10% subsample was processed using the agar-gel method (Slotved et al., 1996). A 10% sample of the total contents from the large intestine of pigs in Group B was examined similarly. Residing worms (immature stages) were recovered from the walls of the large intestine as described by Christensen et al. (1995). All the samples were fixed and stored in an iodine solution (6.25% iodine, 31.25% potassium iodine and 62.5% distilled water). Immediately before counting, the samples were decolorized with a 3% thiosulphate solution which leaves the fluid clean but the worms coloured. The different developmental stages of O. dentatum were differentiated according to the morphology of the buccal capsule (Goodey, 1926). The sex of the adults of both the species was noted. Up to 10 males and 10 females were randomly selected from each of the five sections of the large intestine from each pig which received a challenge infection of O. dentatum (Group C and D). The lengths of the adult worms were measured using a digital image analysis system (Microvision1, DTI, Denmark). Unfortunately, the A. suum worms were not measured. Swine sera were tested in duplicate against A. suum L2/L3 excretory/secretory (ES) antigens by the use of a direct ELISA technique specific for IgG antibodies modified after

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Lind et al. (1993). The technique is described by Helwigh and Nansen (1998). No O. dentatum ELISA technique was available. 2.5. Calculations and statistical analyses All calculations and statistical analyses are based on measurements from the challenge infections. The means were calculated as arithmetic means. The mean length of female and male O. dentatum in each pig was calculated as the mean of the weighted mean lengths from each intestinal section of the pig. Differences in O. dentatum worm burdens and male/female ratios (measured as the total number of males divided by the total number of females) from Group C and D were analysed using Student's t-test. Sample means compared with Mann±Whitney's U-test included: total recovery of A. suum worms and number of white spots on the liver surface log (y ‡ 1) transformed from Group A and B by slaughter day, O. dentatum EPG on Day 18±28 p.c.i., fecundity of O. dentatum (mean EPG at time of slaughter/total female worm burdens per pig), mean length of the O. dentatum worms from Group C and D, and weight gain of the pigs between all the groups and between the male and female pigs within each group. The correlation between the number of worms in the trickle and challenge infections in Group A and C by slaughter day and the correlation between number of O. dentatum worms, length of the worms and EPG in Group C and D were calculated using Pearsons correlation coefficient. The proportions of A. suum worm burdens in liver, lungs and the four sections of the small intestine in Group A and B on the two slaughter days and the proportions of O. dentatum worm burdens in the five sections of the large intestine in Group C and D were compared by repeated-measures analysis of variance to test for the effect of trickle infection. To examine whether changes in A. suum worm burdens and changes in the number of white spots on liver surface between slaughter days 10 and 28 p.c.i. were similar in Group A and B, two-way analysis of variance (ANOVA) was performed on log (y ‡ 1) transformed worm counts and liver spots to test for the effect of trickle infections, slaughter day and their interaction. An ANOVA was also used to test the effect of host sex within the groups on the total worm recovery from the challenge infections. 3. Results 3.1. Performance of pigs Clinical signs of parasitism were not observed in any of the pigs. There were no significant differences in weight gain between groups or between the male and female pigs within groups. The pigs had gained an average of 54.9  5.8 kg at Day 10 p.c.i. and 72.5  5.9 kg at Day 28 p.c.i. 3.2. Trickle infections A. suum worms were recovered from the small intestine in eight of the 12 trickle infected pigs in Group C. Seven of the pigs harboured 20  32 adult worms and one pig

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harboured 20 immature larvae. Macroscopically, the small intestine showed major changes including hypertrophy of the tunica muscularis and the mucosa was very wrinkled and almost dry as described by Stephenson et al. (1980). The livers of all the A. suum infected pigs were fibrotic, with few distinct lesions (white spots). O. dentatum worms were recovered from all 24 trickle infected pigs in Group A with an average of 12926  7129 worms. Twenty-two out of 24 pigs were excreting eggs on the day of slaughter. No larvae were isolated from the intestinal walls, which were edematous, hyperaemic and had numerous nodules. No cross contamination between groups were detected; the A. suum infected control group (Group B) harbored only immature A. suum worms at slaughter and had negative faecal egg counts, the O. dentatum infected control group (Group D) harbored only O. dentatum worms at slaughter and had only O. dentatum positive faecal egg counts. 3.3. Challenge infection The recovery and distribution of the A. suum populations after challenge infection in Group A and B are shown in Table 2. The mean number of A. suum worms recovered 10 days after challenge infection were 399 and 434 for Groups A and B, respectively, and were not statistically different from each other (p ˆ 0.75). Twenty-eight days after challenge, 34 and 137 A. suum worms were recovered from Group A and B, respectively, but the worm burdens were not significantly different (p ˆ 0.13), as the variation within groups was high. The relative location of A. suum worms in the pigs was not influenced by previous O. dentatum trickle infections (p > 0.5). Ten days after challenge infection the pigs in Group A and B harboured more than 70% of the worms in the lungs and the first 25% of the small intestine, while at Day 28 p.c.i. more than 75% of the worms were recovered from the middle 50% of the small intestine. The mean numbers of white spots on the surface of the liver in the A. suum challenge infected pigs are shown in Table 2. There was a significantly higher number of white spots in Group A compared to Group B on Day 10 p.c.i. (p ˆ 0.03). The antibody response against A. suum ES-antigen, measured as optical density (ODvalue), is presented in Fig. 1. The antibody response in Group A were significantly higher compared to Group B from Week 2 p.i. until the first slaughter day (10 days p.c.i.) (p < 0.02). In both Group A and B did the response increase significantly from approximately 2 weeks after challenge infection with A. suum compared to the response at the day of challenge infection (p < 0.005). In Group C the antibody response were between 1.25 and 1.55 OD from Week 4 p.i. throughout the experiment. In Group D the antibody response increased a slightly by Week 11 p.i., however, the antibody response in Group C was significantly higher compared with Group D (p < 0.03) from Week 2 p.i. until termination of the experiment. The recovery and distribution of O. dentatum populations in the challenge infected Group C and D are shown in Table 3. The total recovery of 3493 worms in Group C was lower than Group D where 4927 worms were recovered, but the variability within the groups was high and the difference was not significant (p ˆ0.9). The relative location of O. dentatum in the large intestine did not show any significant difference between the Group C and D (p ˆ 0.6). In both the groups, more than 90% of the O. dentatum

Group

A B A B

No. of pigs

12 12 12 12

Slaughter (day p.c.i.)

Whitespots

10 10 28 28

134  70 70  84 32  35 10  15

All the numbers are given as mean  SD. p.c.i. ± post challenge infection. SI I±IV: Section I±IV of the small intestine. For group designations see Table 1.

Liver

Lung

SI I

SI II

SI III

SI IV

Total

146  79 222  130 24 26

101  78 83  62 11  24 65  101

20  49 10  11 17  35 61  84

0.5  0.9 0.8  1.4 48 49  11

399  130 434  179 34  51 136  183

Number of worms 0 0 0 0

131  66 138  62 0.1  0.3 0

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Table 2 Number of white spots on the liver surface and recovery of Ascaris suum from the six locations of the host

227

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Fig. 1. Antibody response against L2/L3 A. suum specific IgG antigen.!: challenge infection. For group designations see Table 1.

population was found in the first two sections of the colon. The average length of male and female O. dentatum worms is presented in Table 3. Both male and female O. dentatum were significantly shorter in the pigs in Group C compared with Group D (p < 0.0001). The O. dentatum male/female ratios in the Groups C and D were 0.95 and 0.97, respectively, and less than 1% of the recovered worms in both groups were pre-adult (L3 or L4 larvae). The mean faecal O. dentatum egg counts in Group C and D challenge infected with O. dentatum are shown in Fig. 2. In both the groups, egg excretion started after Day 15 p.c.i., and all the pigs except one were excreting eggs on Day 23. At each sampling day, the mean EPG was lower in Group C than the control Group D, though there were no significant differences between the groups (p > 0.25). The fecundity (EPG per female at the day of slaughter) of O. dentatum was also slightly lower in Group C than in the

Fig. 2. Mean Oesophagostomum dentatum EPG from Day 15 after challenge infection. For group designations see Table 1.

Group

C D

No. of pigs

Slaughter (day p.c.i.)

12 12

28 28

Cae

Co I

Co II

Co III

Co IV

Total

928  804 424  652

188  211 158  445

3493  1706 8.2  1.0 4927  1783 13.7  2.2

Number of worms 11  15 87  89

All the numbers are given as average  SD. p.c.i. ± post challenge infection. Cae: Caecum, Co 1±4: Section 1±4 of the colon. For group designations see Table 1.

779  950 1586  934 2186  1389 2138  962

Female length (mm)

Male length (mm) 5.7  2.0 10.2  1.4

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Table 3 Recovery of Oesophagostomum dentatum from the five sections of the large intestine and averge length of the female and male worms

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challenge control Group D (2.8  2.7 and 4.2  4.2, respectively) but there was no statistical difference between the groups (p ˆ 0.25). There was no correlation between the recovery of A. suum and O. dentatum in Group A and C infected with both the species (p > 0.3). No correlation was found between the number of O. dentatum recovered, the length of O. dentatum worms and O. dentatum EPG in Group C and D (p > 0.7). Within each group, host sex had no influence on the numbers of A. suum or O. dentatum recovered. 4. Discussion A. suum and O. dentatum are both nematodes and their adult stages live in different parts of the intestinal tract of their hosts. The A. suum larvae migrate from the large intestine (Murrell et al., 1997) through the liver and lungs and back to the lumen of the small intestine where they develop into adult worms. In contrast, O. dentatum has no migratory phase within the host but develops in the wall of the large intestine. The predilection site of O. dentatum is the anterior part of the colon (Christensen et al., 1995; Roepstorff et al., 1996), which is also the site of penetration of newly-hatched A. suum larvae (Murrell et al., 1997). In order to obtain high physiological and immunological responses against the trickle infections, the pigs in the present study were infected 16 times over a 8 week period before challenge infection. The A. suum trickle infected pigs had a significantly higher antibody response against A. suum ES-antigen from Day 14 p.i. until termination of the experiment compared to the other groups. This correlates well with observations made by Lind et al. (1993), who showed elevated-specific immune responses (IgG/IgA) in an experiment where they trickle infected pigs with 500 A. suum eggs twice weekly for 10±16 weeks. In the present experiment the majority of the recovered A. suum worms from Group C were adults, indicating that most of the larvae from the subsequent trickle infections were inhibited in their migration. Eriksen et al. (1992) and Urban et al. (1988) discussed a possible pre-hepatic barrier as a result of trickle infections that inhibits the migration of A. suum larvae. In the O. dentatum trickle infected group the wall of the large intestine was severely changed and faecal egg counts were low and variable. Furthermore, some antibody reaction against A. suum ES-antigen was observed. The O. dentatum worm population consisted of a mixture of juvenile and adult worms. Indicating that larvae from consecutive trickle inoculations were able to establish infection. In the present study the O. dentatum worms in the A. suum trickle infected group were significantly shorter than in the challenge control group, and O. dentatum egg counts were reduced, though not significantly. This suggest an interaction which might be due to the host immune response as sera from O. dentatum trickle infected pigs cross-reacted with A. suum antigen. This is supported by the observations of Lind et al. (1991) infected with O. dentatum and A. suum L2/L3 ES-antigen, however, when they tested pigs naturally infected with O. dentatum from Finland no cross-reactivity was observed. In the present experiment pigs commenced egg excretion about the same time as the control pigs, and thus the interaction did not change the prepatent period of the O. dentatum infection. This is in agreement with Matusevicius (1981), who recovered the same

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number of O. dentatum worms and observed the same prepatent period in pigs that were infected with 2000 O. dentatum and pigs infected with 2000 O. dentatum and 1000 A. suum. However, interactions between O. dentatum and more closely related species have been observed. Christensen et al. (1997) reported an effect of Oesophagostomum quadrispinulatum on the overall recovery, location and in some cases also the egg production of O. dentatum and argued that the reason for the interaction could be due to either host reactions or competition between the two species as they have overlapping predilection sites in the large intestine. The number of white spots on the surface of the liver in the A. suum challenge infected pigs was significantly higher in the O. dentatum trickle infected group than in the challenge control group on day 10 p.c.i. This indicates a stronger host immune response towards migrating A. suum larvae in the livers of the O. dentatum trickle infected pigs and is supported by the observation of cross reactivity between sera from O. dentatum trickle infected pigs and A. suum antigen before challenge infection with A. suum. Larsen et al. (1997) also found sera from A. suum infected pigs to cross-react with O. dentatum antigen. However, in the present study further migration of larvae was not inhibited as there were no differences in the relative location of A. suum worms or the total A. suum worm burdens between the groups. This is in agreement with Matusevicius (1981), who observed no difference in recovery of A. suum worms in pigs 70 days after infection with 1000 A. suum eggs or 1000 A. suum eggs and 2000 O. dentatum larvae, respectively. The pigs did not show any clinical signs of infection or differences in weight gain during the experiment. O. dentatum is generally considered to be a parasite of low pathogenicity (Stewart and Gasbarre, 1989), although Stockdale (1970) observed major pathological changes in the tunica muscularis and on the tunica mucosa at infection levels of 250 000±1 million larvae. Roepstorff et al. (1996) showed a slight decrease in weight gain and food intake in pigs with high levels of O. dentatum infection. The A. suum trickle infected pigs showed liver granulomas and lymphonodules as described by RoneÂus (1966), and changes of the tunica muscularis and tunica mucosa of the small intestine as described by Stephenson et al. (1980). In summary, the A. suum trickle infection resulted in a weak antagonistic interaction on O. dentatum. The O. dentatum worms were significantly shorter and had lower faecal egg counts and fecundity in the A. suum trickle infected group compared to the challenge control group, however, no difference in the prepatent period was observed between the two groups. These changes may result in lower egg-producing capacity or a shorter lifespan of the O. dentatum worms. It would be interesting to investigate the viability of the O. dentatum eggs from A. suum trickle infected pigs and to study a concurrent infection over a longer period. The higher number of white spots on the surface of the liver demonstrated a much stronger host reaction against migrating A. suum larvae when the pigs were first trickle infected with O. dentatum. This observation was correlated with a higher antibody response against A. suum antigen in the pigs infected with both O. dentatum and A. suum compared to the pigs infected with A. suum only. Further investigations of the different immunological parameters of the pigs might elucidate why this reaction is activated and what effect is has on the pig host.

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