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Keywords:

  • environmental health;
  • microbial contamination;
  • Salmonella;
  • soil;
  • veterinary

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aim:  To investigate the inactivation of Salmonella enterica serovar Typhimurium and the faecal indicator Enterococcus faecalis in horse manure:soil mixtures by application of hydrated lime (Ca(OH)2).

Methods and Results:  In laboratory incubations, the inhibitory effect of different concentrations of Ca(OH)2, as well as different application techniques, was tested. Other variables were horse manure:soil ratio, incubation temperature (6 and 14°C) and soil type (sand/clay). Bacterial enumeration by the plate count method in samples taken at increasing intervals revealed that Ca(OH)2 effectively reduced Salmonella Typhimurium numbers. However, to achieve a sufficient reduction, the Ca(OH)2 had to be applied at a sufficient rate, and the amount required varied because of manure:soil ratio and incubation temperature. The results showed that a pH above 11 was needed and that a high pH had to be maintained for up to 7 days. An appropriate application technique for the Ca(OH)2 was also important, so that a high pH was obtained throughout the whole material to be treated. In addition, a high manure:soil ratio in combination with a higher incubation temperature was found to rapidly neutralize the pH and to increase the risk of Salmonella re-growth.

Conclusions:  Application of Ca(OH)2 can be an efficient method for treating a Salmonella-contaminated horse paddock. A high pH is a key factor in Salmonella inactivation, and thus, monitoring the pH during the treatment period is necessary. To avoid re-growth excess manure should be removed for separate treatment elsewhere.

Significance and Impact of the Study:  Persistence of Salmonella in horse paddocks poses a risk of disease transmission to healthy animals and people who come into contact with these animals. An efficient method to de-contaminate a Salmonella-contaminated soil would be a valuable tool for animal welfare and for public health.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Salmonella infection among horses is an emerging concern, in Sweden often associated with international and national transportation of horses for breeding, trade and competition. It is also a common complication among horses receiving antibiotic therapy at animal hospitals (Ward et al. 2005; Singh et al. 2007). Salmonella infection can cause disease, chronic suffering and mortality among foals, whereas adult horses often only have subclinical symptoms with intermittent shedding of Salmonella in faeces (Clarke and Gyles 1993). As a consequence, horse paddocks can be subjected to contaminated manure and accumulated manure heaps can easily become reservoirs of the infection. As Salmonella infection is zoonotic and horses often come into close contact with people, an outbreak among horses can pose a risk to public health (Sanchez et al. 2002).

Salmonella persisting in the environment of a paddock can cause spread of the infection (Jensen et al. 2006). To avoid further disease transmission, it is therefore important to isolate infected animals and to keep contaminated paddocks empty until they can be declared free from Salmonella. Salmonella has been shown to survive in manure and in soil fouled with manure for long periods ranging from approximately 30 days up to a year (Islam et al. 2004; You et al. 2006; Sinton et al. 2007; Nyberg et al. 2010). In addition, persistence in high numbers for up to 35 days in vegetation has been reported (Åström et al. 2006). However, there is limited knowledge of the persistence of Salmonella in soil fouled with horse manure specifically or of how pathogen-contaminated soil should be treated to limit disease transmission.

In Sweden, animal welfare regulations state that all horses must have access to the outdoors for a certain proportion of each day. However, many commercial stables have limited access to land, which causes difficulties if quarantine periods are required for paddocks. Therefore, there is a need for efficient and rapid methods for treating contaminated paddocks. A common method of inactivating pathogenic micro-organisms in manure and sewage biosolids is by application of hydrated lime (calcium hydroxide, Ca(OH)2) (Bennet et al. 2003; Wong and Selvam 2009; National Lime Association, 2010). Hydrated lime can easily increase the pH up to 12 in such materials, which effectively destroys bacterial cell membranes and inhibits pathogens. This has been demonstrated recently in biosolids and poultry manure, in which faecal coliforms and Salmonella were undetectable after 2 h and less than 24 h, respectively, at pH 12 following lime application (Bennet et al. 2003; Bean et al. 2007). It should also be possible to use hydrated lime for the treatment of pathogen-contaminated soil. In contrast to biosolids and manure, soil is generally a much drier and more heterogeneous material, which makes it difficult to draw conclusions regarding treatment of soil from studies performed on wetter materials.

The aim of the present study was to investigate the inactivation of Salmonella Typhimurium and the faecal indicator Enterococcus faecalis in soil contaminated with horse manure. In a laboratory study, the effects of different concentrations of lime and different techniques in terms of lime application were tested. In addition, the effects of horse manure:soil ratio, incubation temperature and soil type on pathogen inactivation were determined. The study was performed under controlled conditions using conventional culture techniques.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Soil and manure collection and preparation of inocula

Two types of agricultural topsoil were collected from locations close to the city of Uppsala, Sweden. One was a sandy soil with pH 6·6, total-C 0·63% and total-N 0·2%, and the other a clay soil with pH 8·0, total-C 1·46% and total-N 0·24%. The soils were collected in April 2008 and were kept frozen at −20°C until use in the study. Fresh horse manure consisting of a pooled faeces sample from healthy horses was collected at the start of the study from a nearby equestrian centre and kept at 6°C for a maximum period of 2 weeks prior to use in the study. The manure was tested for Salmonella with the methods described below to ensure that there was no indigenous Salmonella present. As could be expected, there were high numbers of indigenous Ent. faecalis present in the manure.

Inocula of Salm. Typhimurium (an environmental isolate from horse faeces isolated at the National Veterinary Institute (SVA) in 2008) and Ent. faecalis (ATCC™ 29212) were prepared from stock cultures kept at SVA, by streaking on horse blood agar followed by incubation at 37°C for 24 h. Colony material was thereafter inoculated into Nutrient Broth (Oxoid, Basingstoke, UK) and incubated overnight at 37°C to a cell density of approximately 108 colony-forming units (CFU) ml−1. At the start of each experiment, fresh horse manure was inoculated with Salm. Typhimurium and Ent. faecalis to approximately 107 CFU g−1 manure. Mixing was performed by hand in plastic bags.

Inactivation study

Freshly inoculated horse manure was mixed with 10, 50 or 90% (w/w) sand or clay soil, to create high (90% manure), medium (50% manure) or low (10% manure) manure:soil combinations (Table 1). Then 0, 1 or 2% of Ca(OH)2 was added to these combinations, and they were thoroughly mixed by hand in plastic bags. At field scale, the 1 and 2% rates represent addition of approximately 0·5 and 1 kg Ca(OH)2 m−2 soil, respectively, assuming incorporation to 5 cm depth. The manure:soil combinations were then portioned into separate stomacher bags for each soil type, manure:soil combination, lime concentration and temperature studied, giving a total of 36 bags. The bags were loosely sealed to allow for aeration and kept at 6 or 14°C. Samples for bacterial enumeration and pH measurements were taken on days 0, 1, 3, 7, 14 and 28. The pH was measured in de-ionized water (1 : 10).

Table 1.   Characteristics of the different manure:soil combinations at the start of the study
SoilManure (%)DM* (%)TS† (%)NH4-N (%)Org-N (%)Total N (%)Total C (%)C/N
  1. *Dry matter.

  2. †Total solids.

Sand9026·814·20·052·00·215·728·1
5059·451·10·051·90·204·221·5
1090·587·20·010·90·101·313·7
Clay9031·018·80·082·90·306·421·3
5051·641·70·042·60·274·817·9
1074·068·30·012·40·252·28·8

To determine the effects of Ca(OH)2 application techniques, fresh inoculated horse manure was mixed with 50% (w/w) of sand or clay soil to create a medium (50% manure) combination (Table 1). The manure : soil mixtures were then packed into 50-ml falcon tubes (3 cm in diameter) with predrilled holes in the bottom to allow for drainage. A total of 30 tubes were prepared for each soil type, allowing three Ca(OH)2 application techniques, two lime concentrations and five samplings per soil type to be performed. The Ca(OH)2 was applied to the falcon tubes by (i) mixing of 0·6 and 1% of Ca(OH)2 (Mixing), (ii) applying 0·6 and 1% of Ca(OH)2 to the top of the columns, followed by drop-wise addition of 15 ml of water (Watering) or (iii) applying 0·6 and 1% of Ca(OH)2 in the form of milk of lime (18% w/w; lime/water) to the top of the columns (Milk lime). After application of Ca(OH)2, the tubes were kept at 6°C for 15 days, and destructive sampling was performed on days 0, 2, 6, 9 and 15. On each sampling occasion, the content of one falcon tube per treatment was emptied into a plastic bag and mixed. Thereafter, bacterial enumeration was performed. The pH of the mixtures was measured in de-ionized water (1 : 10) on days 2, 6 and 15.

Bacterial enumeration

Bacterial enumeration was performed by the plate count method, as described by Nyberg et al. (2010). In brief, aliquots of 3 g were weighed into cultivation tubes, followed by serial dilutions (1 : 10) in phosphate buffer (M15 pH 7·2; supplied by SVA). From the appropriate dilutions, 0·1-ml portions were spread on xylose lysine desoxycholate (XLD) agar with 0·15% natrium-novobiocin (Oxoid) for detection of Salm. Typhimurium and on Slanetz-Bartley (SlaBa) agar (Oxoid) for detection of Ent. faecalis. The XLD plates were incubated at 37°C for 21 ± 3 h and the SlaBa plates at 45°C for 44 ± 4 h. After incubation, the colonies were counted, and the results expressed as log10 transformed mean values (n = 2) of CFU g−1 soil.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

pH measurements

Total mixing with 1% of Ca(OH)2 raised the pH from 7·6 and 7·8 to 11·5 and 11·3 in the manure:sandy soil and manure:clay soil combinations, respectively (Table 2). This high pH was maintained in the fully mixed samples up to day 6, but by day 15, the pH had decreased to 8·9 in both manure:soil combinations. None of the other application techniques produced a pH above 10 except for 1% of Ca(OH)2 applied as milk of lime (on day 2).

Table 2.   Numbers of Salmonella Typhimurium (log10 CFU) and pH value in a medium manure:soil combination (50% manure) measured on days 2, 6 and 15
SoilDayMixed (0·6%)Mixed (1%)Watered (0·6%)Watered (1%)Milk lime (0·6%)Milk lime (1%)
pHCFUpHCFUpHCFUpHCFUpHCFUpHCFU
  1. The control pH was 7·0 for the sandy soil mixture and 7·3 for the clay soil mixture. Mixed = Ca(OH)2 mixed into soil, watered = Ca(OH)2 applied to surface and watered in to soil and milk lime = Ca(OH)2 applied as milk of lime. Numbers in parenthesis represent the amount of Ca(OH)2 added.

Sand29·47·011·53·99·07·08·86·67·97·17·57·1
69·05·811·82·58·36·48·66·48·26·87·66·4
158·85·38·9<28·56·68·96·78·05·77·66·6
Clay29·16·911·3<28·67·59·27·08·47·210·36·6
68·75·810·8<28·56·98·56·88·27·18·06·8
158·75·98·9<28·66·38·87·48·36·08·57·5

The pH measurements in manure:soil combinations showed that application of both 1 and 2% of Ca(OH)2 raised the pH to approximately 12 for all combinations (Fig. 1), with the exception of the high manure:soil combinations at 14°C, in which the pH reached just below 11 after application of 1% of Ca(OH)2 (Fig. 1d). Depending on the amount of manure and the incubation temperature, the time for the pH to decline varied, with a more rapid reduction in the high manure:soil combinations and a slower decline at 6°C compared with 14°C. At 6°C, application of 2% of Ca(OH)2 kept the pH at approximately 12 during the whole study period, with the exception of the high manure : sandy soil combination, whereas at 14°C, a pH of approximately 12 was only maintained in the low manure combinations. With the high and medium manure : soil combinations, the pH decreased to between 8 and 9 after 15 days of incubation for both 1 and 2% of Ca(OH)2 application at 14°C, whereas at 6°C, the pH was only lowered to the same value after the 1% application.

image

Figure 1.  pH-profile of the sand (dashed line) and clay (black line) soil with high (90%), medium (50%) and low (10%) amount of horse manure after addition of 0% (•/○), 1% (▪/□) and 2% (bsl00066/Δ) of Ca(OH)2 at 6°C (a–c) and 14°C (d–f).

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Inactivation as a function of application technique

The plate counts of Salm. Typhimurium and Ent. faecalis in sandy and clay soil after application of 0·6 or 1% Ca(OH)2 by different application techniques showed that the only treatment that was sufficient to reduce Salm. Typhimurium was mixing with 1% of Ca(OH)2 (Table 2). After this treatment, all Salm. Typhimurium was reduced to below the detection limit (<2 log10 CFU g−1 soil) on the first sampling occasion (day 2) in the clay soil and on day 9 in the sandy soil. The same pattern as for Salm. Typhimurium was seen for Ent. faecalis (data not shown). Application of 1% Ca(OH)2 by mixing gave a rapid reduction in Ent. faecalis in the clay soil, in which bacterial numbers fell to below the detection limit by day 2, and a slower reduction in sandy soil, in which low amounts of bacteria were still detectable at the end of the trial (day 15). The other application techniques (watering, application of milk of lime and application of 0·6% Ca(OH)2) proved to be insufficient in reducing either Salm. Typhimurium or Ent. faecalis numbers, as plate counts showed no difference from the untreated controls.

Inactivation as a function of temperature and manure content

Application of 2% of Ca(OH)2 was sufficient to inactivate Salm. Typhimurium within 3 days regardless of soil type, manure content or temperature (Fig. 2). However, in the high manure combination at 14°C, the initial inactivation was followed by re-growth of Salm. Typhimurium as detected on day 7 (Fig. 2d). This re-growth was not quantifiable on sampling days 15 and 28 because of competitive growth of presumptive coliforms, but the presence of Salmonella was confirmed throughout the study period. In the medium and low manure combinations, 1% of Ca(OH)2 was sufficient to inactivate Salm. Typhimurium at 6°C (Figs 2b–c), whereas the same concentration of hydrated lime at 14°C proved insufficient (Figs 2e–f).

image

Figure 2.  Reduction in Salmonella Typhimurium log10 CFU numbers in sand (dashed line) and clay (black line) soil combined with high (90%), medium (50%) and low (10%) amount of horse manure after addition of 0% (•/○), 1% (▪/□) and 2% (bsl00066/Δ) of Ca(OH)2 at 6°C (a–c) and 14°C (d–f). The detection limit was 2 log10 CFU per gram soil.*Indicates samples in which Salm. Typhimurium was further detected, but quantitative detection was not possible.

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Application of 2% of Ca(OH)2 effectively inactivated Ent. faecalis in all manure:soil combinations with the exception of the low manure:sandy soil combination at 14°C (Fig. 3f). In addition, similar re-growth as seen for Salmonella occurred in the high manure:soil combinations at 14°C (Fig. 3d). Application of 1% of Ca(OH)2 was not sufficient to inactivate Ent. faecalis in any of the manure:soil combinations except for in high manure:sandy soil combination at 14°C (Fig. 3d–f).

image

Figure 3.  Reduction in Enterococcus faecalis log10 CFU numbers in sand (dashed line) and clay (black line) soil with high (90%), medium (50%) and low (10%) amount of horse manure after addition of 0% (•/○), 1% (▪/□) and 2% (bsl00066/Δ) of Ca(OH)2 at 6°C (a–c) and 14°C (d–f). The detection limit was 2 log10 CFU per gram soil.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A crucial factor in Salmonella inactivation in a contaminated soil:manure combination is to obtain a substantial rise in pH in the material to be sanitized (Bennet et al. 2003; Bean et al. 2007). The results from the present study indicate that the pH must be 11 to 12 or higher. Application of hydrated lime can easily raise the pH to these levels. However, the hydrated lime must be fully incorporated into the material to be treated, so that the pH is raised sufficiently in all the pathogen-contaminated material. The different techniques for applying hydrated lime to a Salmonella-infected manure:soil combination tested in the present study were chosen as being likely scenarios for a real paddock, considering differences in landscape and access to tractors, etc. Thus, mechanical incorporation, represented by mixing by hand, and incorporation by watering were included. Applying the hydrated lime first and then watering the soil did not achieve sufficient incorporation. Instead the hydrated lime formed a solid layer on top of the soil, with only minor effects on Salmonella numbers or pH in the soil layers below. It was only by mixing that the pH was sufficiently raised in the whole material and thus effectively reduced Salmonella numbers. However, the amount of hydrated lime required for Salmonella inactivation still varied, with influencing factors being incubation temperature and manure:soil ratio.

At the lower incubation temperature (6°C), application of both 1 and 2% of Ca(OH)2 effectively inactivated Salm. Typhimurium, as pathogen numbers were rapidly reduced within the first days of the study. The only exception to this was in the high manure:clay soil combination, where 1% of Ca(OH)2 was not sufficient to reduce Salm. Typhimurium. In this combination, the pH value was rapidly lowered from above 12 to below 9 over the first 3 days of the study, whereas with the high manure:sandy soil, it took 2 weeks to obtain a similar pH decline. This rapid drop in pH is probably the explanation for the insufficient reduction in Salm. Typhimurium numbers. At the higher incubation temperature (14°C), the same concentrations of Ca(OH)2 were not as effective as they were at 6°C, and the impact of amount of manure in relation to soil became apparent. Although a rapid initial reduction in Salm. Typhimurium was seen in all manure:soil combinations, re-growth of both Salmonella and presumptive coliforms occurred in the high manure:soil combination, resulting in bacterial numbers close to those in the untreated control. Bacterial growth is favoured by a higher incubation temperature (Pietikäinen et al. 2005). In addition, bacterial growth is enabled by a rapid drop in pH such as that which occurred in the high and medium hydrated lime combinations. Previous studies on Ca(OH)2 treatment of biosolids and manure for pathogen reduction have often only followed the process for 1 up to 8 days after application (Bennet et al. 2003; Maguire et al. 2006; Bean et al. 2007). However, poststabilization re-growth of Salmonella has also been reported (Wong and Selvam 2009), which confirms that there is a risk of re-growth of Salm. Typhimurium after the initial reduction has occurred, especially in material with high organic matter content.

A key factor in insufficient Salmonella reduction appears to be a rapid drop in pH. The pH profile revealed a more rapid buffering effect in the high manure:soil combinations. This was more pronounced at the higher incubation temperature, where a pH above 11 was only maintained for approximately 5 days after application of 2% of Ca(OH)2 and where 1% of Ca(OH)2 was not sufficient to raise the pH above 11 at all. Conversely, in the low manure:soil combinations, a pH above 11 was maintained for 21 days or more. The exception to this was in the clay soil after the 1% application, where the pH dropped on day 7. This drop in pH was accompanied by an increase in the number of Salmonella. There are several factors that could explain the rapid buffering of the high manure:soil combination observed here. A high content of organic material, as in the high manure treatment, has been shown to increase microbial activity (Marinari et al. 2000) and reduce bacterial attachment to soil (Guber et al. 2005). Bacterial activities such as soil respiration are also reported to be higher at 14°C than at 4°C (Andersson and Nilsson 2001; Pietikäinen et al. 2005). Carbon dioxide is released through the process of heterotrophic respiration, and each molecule of CO2 can neutralize the hydroxide ions from the Ca(OH)2 and thus help buffer the manure:soil mixture. At 14°C, the only applications of hydrated lime that efficiently reduced Salmonella to below the detection limit were those in which a pH value above 11 was maintained for more than 7 days. The data in the present study are not sufficiently comprehensive to give any exact time limits regarding the duration of high pH. However, the results clearly indicated that re-growth can occur if the pH drops within a few days of hydrated lime addition. Therefore, it might be advisable to repeat application of hydrated lime in a material with high buffering capacity to obtain successful Salmonella reduction. Otherwise, removal of excess manure for separate treatment elsewhere, e.g., by thermal composting or ammonia treatment (Vinnerås 2007), prior to application of lime in the paddock would be recommended.

For an equestrian enterprise with limited access to land, it is important to reduce the treatment time for Salmonella-contaminated paddocks. Thus, in the process of eliminating the infection, it will be important not only to raise the pH sufficiently to obtain Salmonella reduction, but also to ensure that the pH is neutralized to such a degree that the paddock can be safely used after Salmonella disinfection. A high pH in soil on which horses are grazed increases the risk of foot and/or mouth lesions in the animals. The time it takes for the pH to normalize is dependent on various factors, including the amount of lime added (Thulander 1982) and the buffering capacity of the soil. Therefore, the amount of lime applied to a soil needs to be balanced to obtain a successful reduction in Salmonella while at the same time limiting the duration of high pH in the soil. A related issue is the level to which a treatment method needs to reduce Salmonella numbers to be considered successful. Salmonella infection among animals usually requires a rather high infectious dose, more than 106 CFU (Mastroeni and Maskell 2006). However, lower doses have been reported in outbreak situations, depending on the ingested food vehicle and the immuno-competence of affected individuals (Blaser and Newman 1982; Wray and Wray 2000; Werber et al. 2005). In the present study, a high initial concentration of Salmonella was used. Thus, in the combinations with rapid inactivation, a reduction from 7 log10 to the detection limit of 2 log10 (a log 5 reduction) was shown in only a few days. According to EU regulations for animal by-products (ABP) category 3 and manure for commercial use, a log 5 reduction is considered a measure of efficient pathogen reduction (1774/2002/EC). The re-growth that was seen in some combinations at the higher temperature indicates that even below the detection limit, there were Salmonella left in the material that could multiply when the conditions changed. However, if a similar log 5 reduction were to occur in a situation with lower initial concentration of Salmonella, there would probably be very low amounts left in the material after treatment, thus minimizing the risk of disease transmission.

To draw further conclusions regarding inactivation of Salmonella under varying outdoor conditions, appropriate application rates and techniques need to be further tested in field studies. Although field studies with pathogens are not easy to perform, this could be circumvented by the use of an indicator organism. Enterococcus faecalis is commonly used as an indicator organism for faecal pollution (Bitton 1999), and in the present study, the pattern of Ent. faecalis inactivation was similar to that of Salmonella. However, Ent. faecalis was more resistant to lime application than Salmonella, which is in agreement with previous studies showing that Ent. faecalis is more tolerant to various treatments, such as urea (Nordin et al. 2009) and ammonia (Vinnerås et al. 2008).

In conclusion, persistence of Salmonella in horse paddocks poses a risk of disease transmission to healthy animals, both domestic and wild, and to people who come into contact with these animals (Jensen et al. 2006; Skov et al. 2008). The present study showed that treatment with hydrated lime effectively eliminated Salm. Typhimurium in a pathogen-contaminated horse manure:soil mixture. However, the amount of lime required for Salmonella inactivation was influenced by several factors, including the temperature at which the manure:soil combinations were incubated and the amount of manure in relation to soil. In combinations with a high amount of organic matter rapid buffering occurred, which posed a risk of Salmonella re-growth at the higher temperature studied (14°C). Therefore, the recommended strategy for efficient Salmonella reduction in a contaminated horse paddock is to monitor the pH over the time of treatment, thereby offering the possibility of repeated hydrated lime application if a drop in pH is observed. In addition, removal of excess manure for separate treatment elsewhere, prior to application of lime in the paddock, is recommended.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Maria Ehrenberg is gratefully acknowledged for assisting with the laboratory work. The project was funded by the Swedish-Norwegian Foundation for Equine Research and the Swedish Civil Contingencies Agency (MSB).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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