Temperature effect on high salinity depuration of Vibrio vulnificus and V. parahaemolyticus from the Eastern oyster (Crassostrea virginica)

International Journal of Food Microbiology 192 (2015) 66–71

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Temperature effect on high salinity depuration of Vibrio vulnificus and V. parahaemolyticus from the Eastern oyster (Crassostrea virginica) A.M. Larsen a, F.S. Rikard b, W.C. Walton b, C.R. Arias a,⁎ a b

Aquatic Microbiology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, 203 Swingle Hall, Auburn University, Auburn, AL 36849, USA Auburn University Shellfish Laboratory, 150 Agassiz Street, Dauphin Island, AL 36528, USA

a r t i c l e

i n f o

Article history: Received 1 July 2014 Received in revised form 21 September 2014 Accepted 24 September 2014 Available online 2 October 2014 Keywords: Vibrio vulnificus Vibrio parahaemolyticus Crassostrea virginica Depuration Salinity Temperature

a b s t r a c t Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) are opportunistic human pathogens naturally associated with the Eastern oyster Crassostrea virginica. The abundances of both pathogens in oysters are positively correlated with temperature, thus ingestion of raw oysters during the warm summer months is a risk factor for contracting illness from these bacteria. Current post-harvest processing (PHP) methods for elimination of these pathogens are expensive and kill the oyster, changing their organoleptic properties and making them less appealing to some consumers. High salinity has proven effective in reducing Vv numbers in the wild and our research aims at developing an indoor recirculating system to reduce pathogenic Vibrios while maintaining the taste and texture of live oysters. The goal of this study was to determine the influence of temperature on the efficacy of high salinity depuration. Vv was enumerated as most probable number (MPN) per gram of oyster tissue using the FDA-approved modified cellobiose polymyxin colistin (mCPC) protocol and with an alternative Vibrio specific media CHROMagar™ Vibrio (CaV). CaV was also used to quantify Vp. Oysters were held at 35 psu for 10 days at three temperatures: low (20 °C), mid (22.5 °C) and high (25 °C). There was no difference in MPN/g of Vv between media; however more Vv isolates were obtained from mCPC than CaV. There was no significant effect of temperature on reduction of Vv or Vp throughout depuration but there was a tendency for low temperatures to be less effective than the higher ones. High salinity resulted in a significant decrease in Vv by day 3 and again by day 10, and a decrease in Vp by day 3. Oyster condition indices were maintained throughout depuration and mortality was low (4% across three trials). Overall these results support the use of mCPC for Vv enumeration and demonstrate the promise of high salinity depuration for PHP of the Eastern oyster. The trend for lower temperatures to be less effective is surprising and indicates a potential interaction between salinity and temperature that should be further investigated. © 2014 Published by Elsevier B.V.

1. Introduction The Gulf of Mexico coast supports a booming oyster industry, accounting for more than 86% of the United States' commercial landings with a value of nearly $75 million (NMFS, 2012). The consumption of raw oysters, particularly those harvested from the Gulf in warmer months, is strongly correlated with serious foodborne illness caused by the Gram-negative bacterium Vibrio vulnificus (Vv; Shapiro et al., 1998). Although foodborne illnesses attributed to Vv are rare (~0.04 illnesses per 100,000 people; Newton et al., 2012), this bacterium is the leading cause of seafood-related deaths in the United States (Oliver, 2013). The seriousness of this illness is due to its high mortality rate

Abbreviations: Vv, Vibrio vulnificus; Vp, Vibrio parahaemolyticus; CaV, CHROMagar™ Vibrio; PHP, post-harvest processing. ⁎ Corresponding author. Tel.: +1 334 844 9215. E-mail addresses: [email protected] (A.M. Larsen), [email protected] (F.S. Rikard), [email protected] (W.C. Walton), [email protected] (C.R. Arias).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.025 0168-1605/© 2014 Published by Elsevier B.V.

(up to 50%) in patients with bloodstream infections, a condition known as primary septicemia (Oliver, 2013). Nearly all cases of primary septicemia occur in patients with preexisting medical conditions, often liver disease (Shapiro et al., 1998). Despite coordinated efforts by the U.S. Food and Drug Administration (FDA) and the Interstate Shellfish Sanitation Conference (ISSC), restrictions regarding Gulf oyster harvesting and refrigeration times have not satisfactorily reduced Vv illnesses (ISSC, 2011). As a result in 2009, the FDA announced its intention to require post-harvest processing (PHP) of Gulf oysters intended for the raw market that are harvested during the warm months. However questions regarding feasibility from the industry and local governments led to an independent assessment on the proposed regulation including cost of PHP, economic impact, and consumer approval (Muth et al., 2011). The results indicated higher costs associated with PHP that would lead to an increase in the cost of Gulf oysters. Further, PHP may influence the taste and texture of oysters which may impact consumer demand (Bruner et al., 2011; Gulf States Marine Fisheries Commission, 2012; Otwell et al., 2011). Thus in order to make PHP more economically favorable, new PHP

A.M. Larsen et al. / International Journal of Food Microbiology 192 (2015) 66–71

methods are under examination that would maintain the organoleptic properties of live raw oysters while producing a product safe for consumers. High salinity treatment of oysters is one new method under investigation as previous research indicates a significant decrease in Vv concentrations in live oysters held at high salinities (Larsen et al., 2013; Motes and DePaola, 1996). Our group previously demonstrated effective reduction of Vv numbers to non-detectable levels (b30 most probable number per gram, MPN/g, as recommended by the FDA) in 50% of trials where oysters were held at 35 psu in an indoor recirculating system (Larsen et al., 2013). Vv numbers are also known to be positively correlated with water temperature (Drake et al., 2007; Kelly and Dinuzzo, 1985) and it is possible that reducing the tank temperature during depuration will increase the efficacy. In addition to Vv, raw oyster consumption is also a risk factor for another foodborne pathogen, Vibrio parahaemolyticus (Vp), the leading cause of seafood-borne gastroenteritis in the United States (Depaola et al., 1990). Vp numbers are also influenced by water temperature and salinity (DePaola et al., 1990, 2003; Johnson et al., 2010; Zimmerman et al., 2007) and as a result abundances of this pathogen may also be impacted by depuration procedures. Some studies have considered Vp in depuration experiments with the Eastern oyster in the wild (Audemard et al., 2011; Walton et al., 2013), but studies in recirculating systems have only focused on temperature effects (Chae et al., 2009; Lopez-Joven et al., 2011). A variety of selective media are available for detection of Vv and Vp in environmental samples. The FDA-approved protocol calls for the use of thiosulfate citrate bile salts sucrose (TCBS) for enumeration of Vp and either modified cellobiose polymyxin colistin (mCPC) or cellobiose colistin (CC) for detection of Vv. However, a number of studies have demonstrated increased sensitivity and specificity of chromogenic agars such as CHROMagar™ Vibrio (CaV) for detection of Vp in seafood as compared to TCBS (Blanco-Abad et al., 2009; Canizalez-Roman et al., 2011; Di Pinto et al., 2011; Duan and Su, 2005; Hara-Kudo et al., 2001; Hassan et al., 2012; Messelhausser et al., 2010). CaV has also proven superior against TCBS and CC for detection of Vv (Cruz et al., 2013; Mahmud et al., 2010; Messelhausser et al., 2010; Williams et al., 2013), however no studies have compared it to mCPC. The goals of this study were as follows: 1) test the efficacy of a high salinity depuration system at three different water temperatures, 2) compare a commercially available chromogenic agar (CaV) for Vibrio spp. with the FDA-approved mCPC methodology, and 3) determine the effect of high salinity depuration on Vp.

2. Materials and methods 2.1. Oyster stock Oysters used in this study were hatchery-reared seed grown in 12 mm mesh bags within an OysterGro cage (Bouctouche Bay Industries Ltd., Bouctouche, New Brunswick, Canada) on an oyster farm in Grand Bay, Alabama, USA. Oysters were approximately two years old at the time of the trials. The day prior to the start of a trial, 300 oysters were removed from the farm and transferred to the Auburn University Shellfish Laboratory. Oysters were scraped free of fouling, rinsed, and left at ambient temperature (ca. 30 °C) overnight (18 h) to increase naturally occurring pathogenic Vibrio numbers. As per FDA guidelines, evaluation of post-harvest protocols requires an initial Vibrio load of 10,000 MPN/g of oyster tissue (FDA, 2011). Due to varying survival during temperature abuse, depuration tanks were stocked with 81 oysters per tank for trials 1 & 2 and 92 oysters per tank for trial 3. Twelve oysters were removed at the beginning of each trial (following temperature abuse but prior to depuration) and from each temperature treatment at the end of each trial for calculation of condition index (CI). Oysters were frozen at −20 °C until CI analysis.

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2.2. Depuration system The recirculating depuration system used in this study was identical to that from Larsen et al. (2013). Artificial seawater made with dechlorinated tap water and artificial sea salt (Instant Ocean®, United Pet Brand Inc., Cincinnati, OH) moved from a 680 L holding tank through three 5 μm particle filters (Aquatic Ecosystems, Inc., Apopka, FL) followed by a 240 W 6-tube UV sterilizer (Aqua Ultraviolet, Temecula, CA). Water then moved into a 360 L raceway tank where oysters were suspended 12 cm from the bottom of the tank in a monolayer on a mesh (2.5 cm) tray. All tanks were maintained at a salinity of 35 psu with varying temperatures. To reach the desired temperatures, the depuration room was lowered to 19 °C. Three 500 W titanium heaters (TH-500, Finnex, IL, USA) attached to heater controllers (HC-0800, Finnex, IL, USA) were added to each tank with one heater–controller combination at both the front and back of the raceway tanks, as well as one in the holding tank. Three temperatures (20 °C, 22.5 °C, and 25 °C) were tested in each of three independent trials (1–3) conducted in late summer of 2012. Depuration trials lasted for 10 days during which temperature and salinity were checked twice daily using a YSI model 85 probe (YSI Inc., Yellow Springs, OH, USA). Any dead oysters were removed from the systems daily. To prevent starvation and potential reduction in oyster condition during the course of the trial, oysters were fed Instant Algae® Shellfish Diet 1800 (ReedMariculture Inc., Campbell, CA, USA) at a volume of 0.25 mL/oyster each morning. During feeding, the system's flow was diverted from the particle filters for 1 h. Particle filters were cleaned every 2 days. 2.3. Condition index CI was calculated using the equation by Lawrence and Scott (1982). After thawing, whole wet weight (WWW) was measured. Meat was removed and dried at 80 °C for 48 h, followed by a measurement of dry tissue weight (DTW). During this time, remaining shells were air dried. After 48 h, dry shell weight (DSW) was measured. CI was calculated as follows: CI = DTW / (WWW − DSW) × 100. All measurements were made in grams to the nearest 0.01 g. 2.4. Vibrio enumeration At each sampling point (day 0, prior to depuration but after temperature abuse, and days 3, 6, and 10), loads of Vv and Vp were determined according to the 3-tube Most Probable Number (MPN) method as described in the FDA Bacteriological Analytical Manual (BAM; Kaysner and DePaola, 2004). Twelve oysters were used at each sampling point to create a homogenate that was diluted, enriched overnight, and plated onto each medium tested. Enrichments were plated on mCPC and CaV (CHROMagar, Paris, France) to compare the recovery efficacy of Vv on CaV versus mCPC (FDA recommended medium). As CaV has previously been demonstrated to better detect Vp in seafood samples, CaV was used in place of TCBS in the MPN procedure for Vp. Following the MPN procedure using mCPC and CaV, colony lift and colony blot hybridization were used to positively identify the isolates. Species-specific oligonucleotide probes (DNA Technology, Aarhus, Denmark) targeting the thermostable direct hemolysin (tdh) and the cytolysin (vvh) genes of Vp and Vv were used to confirm putative colonies as per Takeda (1983) and Wright et al. (1993), respectively. All numbers are given as MPN/g of oyster tissue. 2.5. Data analysis Statistics were performed using XLStat version 2013 (Addinsoft, Paris, France). Statistical analysis was performed on percent change in MPN/g from day 0 in all cases (% change = [MPN/g at day x − MPN/g at day 0] / [MPN/g at day 0]). Data were rank transformed to meet the assumptions of normality and homogeneity of variance. A three-way

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45

ANOVA followed by Tukey's HSD post-hoc test was used to identify the effects of temperature, day, and media on the change in MPN/g of Vv. The effects of temperature and day on the change in MPN/g of Vp were determined by a two-way ANOVA followed by Tukey's HSD post-hoc test. A one-way ANOVA was used to examine the effects of temperature on condition index and a two-way ANOVA was used to determine the effects of temperature and day on mortality. All ANOVAs were considered significant at p b 0.05. Differences between initial and final CI were investigated using a Student's t test (α = 0.01).

Average MPN/g (x1000)

40

3. Results and discussion

35 30 25 20 15 10 5 0

3.1. Pre-trial data

Low

Tank set-up and temperature controls allowed for stable maintenance of temperature and salinity throughout each trial (Table 1). The mid temperature tank during trial 2 averaged 1 °C higher than the comparable tanks of trials 1 & 3, however the temperatures remained statistically different between low, mid, and high temperatures (data not shown). Vp and Vv starting concentrations were similar and were above 10,000 MPN/g following temperature abuse in all trials except trial 1. There was an increase in day 0 MPN from trial 1 to trial 3 that was not correlated with air temperature during temperature abuse or water quality data prior to harvesting (data not shown). 3.2. Temperature There were no significant interactions between any factors in either the three-way ANOVA (Vv; temperature, day, and media) or two-way ANOVA (Vp; temperature and day). After 10 days of depuration, there was no significant impact of tested temperatures on levels of Vv or Vp (Fig. 1). A number of studies indicate that temperature has a strong influence on Vv (Kaspar and Tamplin, 1993; Motes et al., 1998; O'Neill et al., 1992; Pfeffer et al., 2003; Randa et al., 2004) and Vp abundances (Cook et al., 2002; DePaola et al., 2003; Johnson et al., 2010; Parveen et al., 2008). However Wright et al. (1996) and Zimmerman et al. (2007) found no significant correlation between temperature and Vv and Vp during warm months, respectively. In oysters, numbers of these pathogens seem to stabilize around 25–26 °C (DePaola et al., 2003; Motes et al., 1998). Past studies have seen an interaction between temperature and salinity on survival, with lower temperatures increasing survival at higher salinities (Covert and Woodburn, 1972; Kaspar and Tamplin, 1993). Our data also support this trend, as raw values of Vv were always higher (although not significantly) in low temperature tanks than mid and high temperature tanks. Vv is isolated at lower frequencies after prolonged exposure to high salinities (N 25 psu) (Arias et al., 1999; Larsen et al., 2013; Motes and DePaola, 1996; Motes et al., 1998). The relationship between Vp and salinity is far less understood, but DePaola et al. (2000) detected a negative correlation with salinity from 15 to 29 psu, and Shen et al. (2009) saw a decrease in Vp in oysters after 32 h of exposure to water at 28 psu at a variety of temperatures. Our study suggests that high salinities may reduce the impact of temperature on these species, although larger temperature differences need to be tested to confirm this prediction. Also, a study testing three

Mid

High

Low

Mid

High

Fig. 1. Average MPN/g including standard error for all trials (1–3) at each temperature. Black, V. vulnificus, gray, V. parahaemolyticus.

temperatures and three salinities for example could provide insight into potential interactions between temperature and salinity on Vv and Vp abundances. 3.3. Time course Vv numbers decreased throughout depuration, with significant reductions seen in oysters at day 3 relative to day 0, followed by another significant decrease at day 10 (Table 2). All temperature treatments reached b30 MPN/g in trial 1, as did high and mid temperature tanks in trial 2 (Fig. 2). No treatments reached below 30 MPN/g in trial 3. It is expected that trials with higher initial Vv concentrations resulted in fewer replicates reaching the non-detection limit. Perhaps with longer depuration times, these trials would also have reached the non-detection value. Audemard et al. (2011) saw a decrease in Vv to b30 MPN/g after 7 days in all trials, although starting concentrations were less than 250 MPN/g. Motes and DePaola (1996) successfully depurated Vv from oysters to b10 MPN/g in 7 to 17 days, with one trial with a higher initial concentration taking about 30 days. Over 50% of our tanks experienced an increase in Vv numbers from day 3 to day 6; the reason for this is unknown. Days with a significant decrease were the same as those in our previous experiment (day 3 and 10) indicating a consistency in depuration in our system despite changes in salinity (Larsen et al., 2013) and temperature (this study). Vp numbers also decreased although numbers were only statistically significant between day 0 and day 3 (Table 2). At each time point (except day 0 in trials 2 and 3 which were identical) values of Vp were higher than Vv numbers on the same media. Vp only reached b30 MPN/g at 3 time points during all trials: day 3 in the mid temperature tank and days 3 and 6 in the high temperature tank in trial 1. Chae et al. (2009) analyzed the depuration of both Vv and Vp at 30 psu at a variety of temperatures and found that Vv numbers were significantly reduced faster than Vp. Audemard et al. (2011) saw a decrease in Vp in oysters relayed to high salinity waters although starting concentrations were low (≤75 MPN/g). Thus although there is an apparent effect of salinity on Vp concentrations, this impact does not seem to be as strong as that on Vv.

Table 1 Temperature (°C) and salinity (psu) values ± standard deviation for each tank and each trial. Low

Trial 1 Trial 2 Trial 3

Mid

High

Temperature

Salinity

Temperature

Salinity

Temperature

Salinity

19.5 ± 0.36 20.7 ± 0.45 19.5 ± 0.26

35.1 ± 0.23 34.9 ± 0.41 35.1 ± 0.18

22.3 ± 0.38 23.0 ± 0.15 22.8 ± 0.38

35.0 ± 0.30 35.3 ± 0.33 35.1 ± 0.19

25.0 ± 0.16 25.0 ± 0.28 25.1 ± 0.17

35.3 ± 0.43 35.1 ± 0.32 35.1 ± 0.32

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Table 2 MPN/g results for Vibrio vulnificus (average of mCPC and CHROMagar™ Vibrio results) and V. parahaemolyticus for each sampling day of each trial. As temperature was not significant, numbers represent averages from all three temperature treatments for each trial ∗ day combination. Significance groupings were determined from an ANOVA followed by Tukey's post-hoc analysis on the rank transformed change in MPN/g from day 0. Day

0 3 6 10

V. vulnificus

V. parahaemolyticus

Trial 1

Trial 2

Trial 3

Average

Group

Trial 1

Trial 2

Trial 3

Average

Group

4300 7.9 55.2 8.3

46,000 4691.7 338.2 101.8

110,000 41,683.3 11,533.3 2831.7

53,433 15,461 3975.6 980.6

A B BC C

9300 29.0 162.5 355.3

46,000 8933.3 12,533.3 4094.3

110,000 49,666.7 18,033.3 6560

55,100 19,543 10,243 3669.9

A B B B

3.4. Media

3.5. Condition index and mortality

CaV did not statistically differ from mCPC in terms of detection of Vv from oysters using the MPN procedure (p = 0.371). However, up to 50.8% of putative Vv isolates on mCPC were confirmed by vvh probe while only 47.2% of colonies recovered on CaV tested positive (Fig. 3). It is noteworthy that 50% of the putative colonies obtained on mCPC were not Vv. This percentage was higher than that seen by Staley et al. (2013) and Macian et al. (2000) but much lower than that obtained by Hoi et al. (1998). CaV showed even less specificity and only produced about 2/3 of the isolates as mCPC. The MPN procedure requires only one positive colony from a dilution for that dilution to be positive but each dilution can have up to 3 positive colonies, thus accounting for the difference in detected colonies and lack of difference in MPN. Previous studies have analyzed the use of CaV to confirm results obtained on other media (Cruz et al., 2013; Williams et al., 2011) but this is the first study to analyze it as an alternative to mCPC. Although this study determined that media do not significantly impact final results in terms of MPN of Vv, it is important to highlight that mCPC yielded more Vv colonies than did CaV and thus mCPC is preferred for this type of study.

There was no impact of temperature (p = 0.698) and no significant difference between final and initial CI during depuration at any temperature (Table 3). However, CI of oysters at 25 °C (high temperature treatment) was lower than that from other temperatures after 10 days of depuration and longer depuration times may lead to a significant result. Regardless, the loss of meat quality experienced during previous depuration trials (Larsen et al., 2013) was avoided by feeding the oysters. Heilmayer et al. (2008) saw an interactive effect of temperature and salinity on CI, with lower temperatures maintaining CI at higher salinities which is the same trend we see in this study, although their study had a max salinity of 25 psu. Respiration rates in the Eastern oyster increase dramatically between 20 and 25 °C (Percy et al., 1971) and feeding rates are highest around 25 °C (Galtsoff, 1964), thus oysters held at 25 °C may need a higher quantity of food to maintain CI. In this study, all oysters were fed the same amount (0.25 mL/oyster). Future studies targeting longer depuration times (more than 10 days) may need to increase the amount of feed in those oysters held at higher temperatures in order to maintain CI.

10000

10000

1000

1000

MPN/g

B 100000

MPN/g

A 100000

100

100 10

10

1

1 Day 0

Day 3

Day 6

Day 10

Day 6

Day 10

Day 0

Day 3

Day 6

Day 10

C 1000000 100000

MPN/g

10000 1000 100 10 1 Day 0

Day 3

Fig. 2. Individual trial results for V. vulnificus (Vv) and V. parahaemolyticus (Vp). MPN/g is displayed on a logarithmic scale. Vv numbers represent an average of mCPC and CHROMagar™ Vibrio results. Horizontal line indicates 30 MPN/g. Black, Vv, gray, Vp. Solid lines, low temperature, dotted lines, mid temperature, dashed lines, high temperature.

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Number of Isolates

70

1200

References

1000

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800 600 400 200 0 mCPC

CaV

Negative Isolates

Positive Isolates

Fig. 3. Number of putative V. vulnificus isolates confirmed (positive) or not (negative) by colony blot hybridization. These results show all V. vulnificus isolates tested during the duration of the study.

There was no difference in mortality between temperature treatments and day (p = 0.316). Total mortality for all three trials was 4.1% and was 3.7%, 2.9%, and 5.4% for trials 1, 2, and 3 respectively.

4. Conclusion High salinities reduced both Vv and Vp loads in oysters over 10 days, with both species significantly declining at day 3, and only Vv decreasing again by day 10. High salinities were able to reduce the numbers of both Vv and Vp to b30 MPN/g as recommended by the FDA, but longer depuration times may be necessary to reach this goal consistently in oysters with higher initial concentrations of these bacteria. There was no significant difference in Vv or Vp concentrations between the temperatures used in this study although there was a tendency for low temperatures (20 °C) to be less effective than higher ones (22.5 °C and 25 °C). CaV did not differ statistically from mCPC in enumeration of Vv from oysters during depuration in terms of MPN/g. However, mCPC recovered more Vv isolates and thus is the preferred media for depuration. CI was maintained for the full 10 days, and average mortality was 4%. Our data show that reduction of pathogenic Vibrios in oysters is possible by depuration when salinity and temperature are maintained in a controlled, recirculating system. Future studies should investigate the feasibility of scaling up these systems to commercial size.

Acknowledgments This research was funded by the Mississippi-Alabama Sea Grant Consortium (MASGC) grant R/AT-10-NSI “Sea Grant Aquaculture Research Program 2010: Eliminating human-pathogenic Vibrio vulnificus from Gulf Coast Oysters with high salinity depuration.” Andrea wishes to thank Auburn University for her Cell & Molecular Biology summer fellowship.

Table 3 Condition index at initial (day 0, post temperature abuse but prior to depuration) and final (day 10) time points. Trial 1 Initial Low Mid High

3.9 4.8 4.4 3.7

± ± ± ±

Trial 2 0.9 1.3 0.8 1.7

5.5 5.7 6.0 5.5

± ± ± ±

Trial 3 1.4 1.2 1.8 1.9

8.4 6.7 6.7 5.2

± ± ± ±

Average 0.8 1.4 1.3 2.5

5.9 5.6 5.7 4.8

± ± ± ±

2.1 1.5 1.7 2.2

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