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Verschuere, L., Rombaut, G., Sorgeloos, P. and Verstraete, W. (2000). Probiotics bacteria as biological control agents in aquaculture. Microbiology and Molecular Biology Review. 64: 655-671.

Studies on alcoholic extract of plantago ovate on some hematological parameters of Oncorhynchus mykiss

Mohammad Javad Mohammadi1*, Mojtaba Alishahi2, Amir Aramon3, Reza Jahantigh4

1 Msc of Islamic Azad University Science and Research Khuzestan, Iran

2 Veterinary faculty, Shahid Chamran University, Ahvaz-Iran

3 Msc of Tehran University - Iran

4 Msc of Urmia University - Iran

* Corresponding author.Email: mohammadimjm@ gmail.com


Pisciculture has benefited human being during last decades. Among different specis Oncorhynchus mykiss is of great important. Its increasing mortality due to infectious diseases and also ineffectivity of synthetic drugs motivated us to evaluate effect of alcoholic extract of plantago ovate on some hematological parameters of this speces. hematological parameters could act as symptoms of health and fitness.

Material and method

This research was done in genetic and breeding research center of cold water fish in Yasouj. Treatments were different doses of alcoholic extract of plantago ovate seeds (0.1, 0.5 and 1 percent). Each treatment was replicated three times and each replicate contained 30 individuals (30 + 5.23 g). Experiments were conducted in a completely randomized design and results were taken and noted done after 60 day feeding.

Results and conclusions

Hematological parameters including hematocrite amount (PCV), red blood cells, median globular volume and median globular hemoglobulin amount were affected significantly at doses 0.5 and 1 percent (p<0.05) while white blood cell number was not affected. All and all it could be

deduced that alcoholic extract could affect hematological parameters in related to red blood cells, hence addition of this extract to daily diet could enhance hematopoiesis.

Key words: Plantago ovata, Oncorhynchus mykiss, Hematological parameters

Effect of Saccharomyces cerevisiae and Lactobacillus casei on disease resistance in rainbow trout (Oncorhynchus mykiss)

Maria Mohseni1*, Amir Tukmechi2, Saeid Meshkini3 and Rezvaneh Jenabi1

1 Department of Fisheries, Faculty of Natural Resources, Urmia University, Iran

2 Department of Pathobiology and Quality Control, Artemia and Aquatic Animals

Research Institute, Urmia University, Iran

3 Department of Food Hygienic and Quality Control, Faculty of Veterinary Medicine,

Urmia University, Iran * Corresponding author: maria.mohseni@gmail.com


Antibiotic use in aquaculture may be detrimental to the environment and human health, and involve the development and transfer of resistance to other aquatic bacteria, fish pathogens and human pathogens, the accumulation of residual antibiotics in aquaculture products and affect the microbial biodiversity (Sharifuzzaman and Austin, 2010). Alternative disease control strategies involve improved husbandry, better nutrition, improvedwater quality and lower stocking densities, and the use of vaccines, beneficial microorganisms (= probiotics) and non specific immunostimulants (Austin and Austin, 2007). The application of probiotics is increasingly used in disease control against bacterial fish pathogens especially in Asia and South America (Brunt and Austin, 2005).In rainbow trout farming, one of the most prominent bacterial diseases is caused by Yersinia ruckeri (Tukmechi et al., 2011). The aim of this study was to investigate the effect of Lactobacillus casei and Saccharomyces cerevisiae as probiotic on disease resistance in rainbow trout (Oncorhynchus mykiss).

Key words: Rainbow Trout, Probiotic, Saccharomyces cerevisiae, Lactobacillus casei, Yersinia ruckeri, bacterial challenge

Materials and Methods

For this purpose, one thousand and fifty fish (25.6±2 g average weight) were obtained from a local fish farm, Urmia, Iran. Healthy fish were kept in 1000 L tanks for 10 days for acclimation to the laboratory conditions. Then, fish were randomly divided into seven groups each with triplicate as follow: 1) control that were fed with commercial pellet, 2) fed with 107 CFU/g Lactobacillus casei, 3) fed with 107 CFU/g Saccharomyces cerevisiae, 4) fed with 107 CFU/g 2ME2 treated Saccharomyces cerevisiae, 5) fed with 10 CFU/g Lactobacillus casei and 107 CFU/g Saccharomyces cerevisiae in combination, 6) fed with 107 CFU/g Lactobacillus casei and 10 CFU/g 2ME treated Saccharomyces cerevisiae in combination and 7) fed with Oxytetracyclin (25 mg/KgBW). The probiotic were sprayed into the diet slowly, and allowed to dry at room temperature for 2 h. For control group only commercially pellet was used without addition any probiotic. 2ME treatment of yeast cells was performed according to Tukmechi et al. (2011). Fifty fish were randomly placed in 1000 L tanks and fed for 45 days. After 45-day feeding trial, 70 fish from each dietary treatment (10 fish per treatment) were obtained, anaesthetized with clove powder (200 mg/L). Then, the fish were challenged by I.P. injection with 0.1 ml of a suspension of Y. ruckeri (BCCM5/LMG3279) (1x107 cells /ml). Dead and moribund fish were removed and examined microbiologically for up to 14 days. More ever, agglutination test was performed on samples for confirmation. All the measurements were made in triplicate. The results were subjected to analysis of variance (ANOVA) followed by least significant differences (Tukey) test. Correlation coefficients were significant with P < 0.05.

Results and discussions

Results showed that addition of L. casei to the diet can improve fish resistance against Y. ruckeri. After 14 days, group 2 (fed with 107 CFU/g

2 beta-mercapto-ethanol

Lactobacillus casei) showed the highest survival rate (64.28 %) that was significantly (p<0.05) higher than all other groups and about 4.5 times more than control group (14.28 %). The lowest survival rate (7.14 %) observed in group 4 (fed with 107 CFU/g 2ME treated Saccharomyces cerevisiae) that was half of control group. Survival rate was respectively higher in group 5 (35.71 %), 6 (28.57 %) and equal in 3 and 7 (21.43 %).

The effectiveness of probiotics in terms of protection against infectious pathogens is often attributed to the elevate immunity. Protection against edwadsiellosis, enteric red mouth disease, furunculosis, lactococossi, streptococcosis and several other diseases are successfully accomplished through probiotics feeding. Furthermore, probiotics treatment leads to better protection of fish from multiple diseases (Nayak, 2010).


Austin B, Austin DA. Bacterial fish pathogens: diseases of farmed and Wild fish. 4th (revised) ed. Godalming: Springer-Praxis; 2007

Brunt J, Austin B. Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 2005;28:693-701.

Nayak, S.K., 2010. Probiotics and immunity: A fish perspective. Fish & Shellfish Immunology, 29(1), 2-14. Available at: http://dx.doi.org/10.1016/j.fsi.2010.02.017.

Sharifuzzaman, S.M. and Austin, B., 2009. Influence of probiotic feeding duration on disease resistance and immune parameters in rainbow trout. Fish and Shellfish Immunology 27(3): 440-445.

Tukmechi, A. et al., 2011. Dietary administration of beta-mercapto-ethanol treated Saccharomyces cerevisiae enhanced the growth, innate immune response and disease resistance of the rainbow trout, Oncorhynchus mykiss. Fish and Shellfish

Immunology, 30(3): 923-928.

Interrelationship Among dietary Methionine and Choline in beluga, Huso huso

Mohseni* M., Pourali H.R., Hassani H. & Yazdani M.A.S.

International Sturgeon Research Institute, P.O. Box 41635-3464, Rasht, Guilan, Iran * Corresponding author: Mahmoud Mohseni mahmoudmohseni@yahoo.com

Background (or Objective) of This Study

When two or more nutrients in a fish diet are interrelated, it is necessary to consider how their presence and quantity will influence the growth of fish and economic benefits of fish production when one or all nutrients are supplemented to the diet. Methionine is an essential amino acid that is required by fish. Choline is considered a vitamin in the diets of vertebrate such as poultry, dogs, and several species of fish. methionine, choline, and betaine share a common metabolic use as methyl donors, there may be an interrelationship among dietary methionine and choline. Therefore, a better understanding of the interrelationship among methionine and choline may not only improve fish growth performance but also reduce the cost of feed. The objective of the present research was to determine if there is an interrelationship between methionine and choline in feed formulations for the beluga.


A 3 x 3 factorial design was used to reevaluate the dietary Methionine requirements and to determine the optimum dietary choline in juvenile beluga. Each diet was randomly fed to triplicate groups of fish (20 fish/each group) over a 10-wk growth trial. Nine isonitrogenous (420 g kg-1), isolipidic (152 g kg-1 diet) and isoenergetic (20 MJ kg-1) diets were formulated and prepared to contain three methionine levels (0.75, 1.5 and 2.25%) and three choline levels (300, 600, and 900 mg/kg diet) at each Methionine level: M0.75Ch300, M0.75Ch600, M0.75Ch900, M1.5Ch300, M1.5Ch600, M1.5Ch900, M2.25Ch300, M2.25Ch600, and M2.25Ch900.

There were significant Methionine and Choline effects, also there

were interactive effects on WG, SGR, FE, and PER (P < 0.05). There

were no significant differences in weight gain, SGR, PER, and FCR, between fish fed M0.75Ch900 (low methionine and high choline) and Mi.5Ch600 (sufficient methionine and sufficient choline). There were no significant differences in the measured parameters between M2.25Ch300 (low choline and high methionine), M1.5Ch600 (median choline and median methionine) and M2.25Ch600 (sufficient choline and high methionine). Methionine had no significant linear effect on weight gain and feed efficiency when choline was low. Therefore results indicated that in the absence of sufficient methionine, choline can spare a portion of methionine requirement of beluga fed semi-purified diets. However, in the absence of sufficient choline, Methionine cannot spare the portion of the choline requirement of beluga fed semi-purified diets.

Protective Effect of Truncated Heat Shock Protein 70s on Artemia franciscana Against Pathogenic Vibrio campbellii challenge

Parisa Norouzitallab*, Kartik Baruah, Patrick Sorgeloos and Peter Bossier

1 Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Rozier

44, 9000 Ghent, Belgium

* E-mail: pnorouzitallab @ gmail.com Introduction

Recent evidences have shown that heat shock protein 70 (Hsp70) can be a possible anti- infective strategy to contain disease in aquaculture (Baruah et al. 2010). Hsp70s are highly immunogenic proteins, with an exceptional degree of evolutionary conservation. Hsp70 derived from either prokaryotes (DnaK) or eukaryotes consist of three functionally distinct domains: an N-terminal 44-kDa ATPase portion, an 18-kDa peptide-binding domain, and a C-terminal 10-kDa fragment. In a recent study, Baruah et al. (2010) compared the efficacy of two Hsp70s derived from either Artemia or E. coli, both of which have a high degree of amino acid sequence homology, in conferring protection to Vibrio-challenge Artemia. They found that Artemia Hsp70 and DnaK are equally efficient in protecting Artemia against Vibrio. Similar protective effect of these proteins could possibly be due to the high degree of homology (59.6%) between them, particularly in the peptide-binding domain (the putative innate immunity-activating portion). This suggests that the observed protective capacity of these proteins might reside within this peptide-binding domain. However, no in vivo studies have been carried out to determine the immunological/protective functions of this domain. This study aimed to verify whether the C-terminal portion (containing the 18-kDa peptide- binding domain) of either Artemia Hsp70 or DnaK could modulate the immune responses of Artemia and confer protection against Vibrio campbellii challenge.

The truncated portion of Artemia Hsp70, Artemia Hsp359-644 (A359) and E. coli Hsp70 or DnaK, E. coli Hsp359-638 (E359) were prepared from the One Shot TOP10 (non-pathogenic E. coli) cells as described previously (Baruah et al., 2010). Overproduction of Hsp70 proteins in the bacterial strains A359 and E359 was determined according to Sung et al. 2009). Axenic Artemia were obtained following procedures described by Baruah et al. (2010). Thirty nauplii were transferred to sterile 50-ml glass tubes that contained 30 ml of autoclaved seawater. The nauplii were incubated for 6 h with the E.


coli strains at 10 cells/ml. They were then challenged with V. campbellii for 36 h, after that Artemia survival was scored. Immunoprobing of the western blot for truncated Artemia Hsp70 and DnaK proteins were carried out as described previously (Sung et al. 2009). Live nauplii from all treatments were harvested after 6 h of Vibrio exposure, frozen in liquid nitrogen and stored at -80 °C for further analysis of phenoloxidase activity by standard protocols. Data were analyzed using ANOVA followed by Duncan's multiple range tests using SPSS version 14.0.


The survival of the non-challenged nauplii fed either induced or non-induced E. coli cells did not differ significantly (P>0.05) between each other (Fig. 1). In contrast, a significant (P<0.001) difference among the challenged groups was noted. Survival of Artemia fed with non-induced strains was low upon challenge with Vibrio. But a significantly (P<0.001) higher survival was obtained when arabinose-induced A359 or E359 strains were provided to challenged nauplii in comparison to challenged nauplii supplied with non-induced E. coli cells. The increase in the survival coincided with an increase in the production of truncated Hsp70s (Fig. 3). Results also revealed that the in absence of Vibrio exposure, no significant (P>0.05) changes in the

phenoloxidase (PO) activity level was observed in Artemia fed with induced or non-induced E. coli strains. However, after exposure to Vibrio, nauplii fed with induced A359 or E359, exhibited a significant increase in the activity of PO (Fig. 2).

Fig. 1. Artemia survival (%) after 36 h challenge with

V. campbellii. * indicates significant differences Fig. 2. Apparent PO activity in Artemia. * indicates

between induced and non-induced bacteria. (-) and significant differences between induced and non-

(+) indicate non-induction and induction with induced bacteria. (-) and (+) indicate non-induction

arabinose, respectively and induction with arabinose, respectively.


M        A359 (-) A359 (+) E359 (-) E359 (+)

Fig. 3. Arabinose-induced expression of A359 and E359 proteins in the A359 and E359 strains, respectively. '-' and '+' indicate non-induction and induction with arabinose at 0.5 mg/ml. Molecular mass standards (M) in kDa are on the left.


Overall results showed that Artemia fed with either A359 or E359 strains overproducing truncated A359 or E359 proteins, respectively, at an optimum arabinose dose of 0.5 mg/ml, showed the best survival in the Vibrio challenge assay. The observed effect was due to enhancement of the Artemia immune system as PO activity

was found to be increased by these proteins, in agreement with the findings of Baruah et al. (2010) who observed similar increase in the activity of PO enzyme in Artemia fed

Hsp70s. References

Baruah K, Ranjan, JK, Sorgeloos P, Bossier P. Efficacy of homologous and heterologous heat shock protein 70s as protective agents to gnotobiotic Artemia franciscana challenged with Vibrio campbellii. Fish Shellfish Immunol 2010;29:733-39.

Sung YY, Dhaene T, Defoirdt T, Boon N, MacRae TH, Sorgeloos P, Bossier P. Ingestion of bacteria overproducing DnaK attenuates Vibrio infection of Artemia franciscana larvae. Cell Stress Chap 2009;14:603-9.

Characterising Artemia biodiversity: the special Urmia case and plea for a gene bank

Peter Bossier1, Ramin Manaffar2, Naser Agh2, Gilbert Van Stappen1, Patrick Sorgeloos1

1 Laboratory of Aquaculture and Artemia Reference Center, University of Ghent, Gent, Belgium

2 Artemia and Aquatic Animals Research Institute, Urmia University, Urmia, Iran

Artemia cysts, originating from a variety of locations, are currently commercially exploited. The overall quality of the cysts depends on a lot of features including their intrinsic nutritional quality, harvesting and processing conditions, diapause characteristics and size, all influencing their commercial value. In order to mediate authentication of commercial Artemia cyst samples, a database containing eight RFLP patterns of a 1500 bp mitochondrial 12S-16S rDNA fragment was constructed. The database contains 53 samples, covering most of the geographical distribution of Artemia. On the basis of a band sharing index these 53 samples could be clustered into five groups. These groups coincide to a great extent with the currently accepted species or species complexes.

Within each cluster diversity between samples is still considerable, reflecting the genetic diversity within each species. The developed method allows assigning samples to these clusters, facilitating their authentication at the species level. This technique allowed demonstrating that A. urmiana is very closely related to parthnogenetic Artemia, despite its specific morphological charateristics.

Using a nuclear marker, more specifically the cDNA of the HSP26 gene, diversity among Artemia was revisited. The data revealed more or less a similar pattern, with the lowest genetic distance between A. urmiana and parthenogenetic Artemia.

In order to find a marker for differentiating between a bisexual and a parthenogenetic Artemia strain, Exon-7 of the NaK ATPase a1 subunit gene was screened by RFLP technique. The results revealed a constant synonymous SNP (single nucleotide polymorphism) in digestion by the

Tru1I enzyme that was consistent with these two types of Artemia. This SNP was identified as an accurate molecular marker for discrimination between bisexual and parthenogenetic Artemia. According to the Nei's genetic distance (1973), the lowest genetic distance was found between individuals from Artemia urmiana and parthenogenetic populations, making the described marker the first marker to easily distinguish between these two co-occurring species.

Given the fluctuations of the lake over geological times, we thus hypothesized that species identification of Artemia cysts, buried in the sediments, can provide information on lake conditions in the past. Therefore, encysted embryos of Artemia were recovered from lake sediments by augering at a site near the present shoreline. Cysts and associated plant remains from two studied levels yielded radiocarbon ages in the range 5000-6700 YBP. For determination of the type of Artemia, the constant synonym mutation in exon-7 of the Na/K ATP-ase gene was verified, and the diameter of the recovered cysts was compared with that of modern cysts from the Lake Urmia region. The results show that the cysts represent a parthenogenetic type of Artemia, whose cyst diameter is somewhat different from that of present-day local parthenogenetic Artemia. The present study firstly confirms the stability of DNA in ancient Artemia cysts for molecular analysis. Moreover, it suggests variation in Lake Urmia's conditions over time, and based on comparison with salinity preferences of contemporary Artemia populations, it more specifically suggests that Lake Urmia was a brackish lake dominated by a parthenogenetic Artemia population in the geological period sampled. It finally illustrates how, like in the study of freshwater propagule banks, paleogenetic analysis of Artemia DNA recovered from sediment cores can be used as a tool in the paleoecological study of generally highly fluctuating saline habitats.

It is clear that the current knowledge on Artemia biodiversity signals a series of very interesting phenomena. Further characterization of this diversity will be helped by the use of proper genetic markers. For instance the use of AFLP markers has allowed to identify sex related markers. Also the ARC-Ugent is currently establishing the Artemia

franciscana genome sequence, which could provide a template for sequencing the A. urmiana genome, further allowing documenting its specific position within Artemia phylogeny.

It is clear that environmental circumstances in lake Urmia are very variable from geological and historical perspectives. Artemia populations are adapting to that. Yet the current speed of change in lake Urmia might not allow the Artemia populations to adapt to the situation. Hence maintaining a gene bank might be of great value with respect to re-introduction of the necessary gene pool, in case lake conditions could be restored quickly.

Effect of various salinity levels on the survival and growth rate in Gavkhuni Artemia population (Artemia urmiana)

Parastoo Razmara1*, Mojtaba Haghjou Jahromi1, Eisa Ebrahimi2

1 Department of Fisheries Sciences, Master student of Natural Resources, Isfahan University of Technology, Isfahan, Iran.

2 Assistant professor of Fisheries Sciences. Faculty of Natural Resources, Isfahan University of Technology, Isfahan, Iran.


Live food are known as one of the most important and valuable nutritional factors in aquatic larviculture and are effective on the maximum aquatic larval survival and growth (1). Today, among the numerous and varied sources of live foods, baby brine shrimp or Artemia are important in aquaculture due to high nutritive value, high digestibility, variety, size, ease of production, salt tolerance, and also used as a drug or vitamine transmitter (2). Artemia naplius is more useful among another Artemia products like decapsulated Artemia, freezed Artemia and Artemia powder (3). They are able to tolerate 40 to 220 ppt salinity (Agh, Noori, 1995). They are appropriate for larval stage because of their necessary nutrient (Agh, Noori, 1997). All particles and micro­organisms floating in the water filtration cycle that are digestible can be considered as an Artemia food. Artemia is nonselective filter feeder (1 to 50|i). Phytoplanktons, bacterias and small food ingredients are appropriate food for Artemia (Coutteau, 1992; Brown et al., 1991). Various factors, including salinity, pH, temperature, oxygen and light are effective factors on growth and hatching rate (4). Hatching method, growth rate and the quantity of notified factors are evaluated in all Artemia species (5). Gavkhoni swamp located in the south-east of Isfahan province and covers 476 square kilometers.

The aim of this study was to identify the effects of various salinity level on the Gavkhoni Artemia (Artemia urmiana) growth and larval survival.

The study was conducted in aquaculture unit in Isfahan University of Technology in August, 2012. The study first started with Artemia collecting from Gavkhuni swamp.Then, they transfer to the sodium hypochloride solution (20 ppt) about 15 minute for disinfection and they were placed in four treatments that contain 30, 35, 40, 45 ppt salinity in three replications. 20 Artemia were depleted in each replication tank. In order to provide standard conditions some physiochemical factors like temperature, pH, photoperiod and aeration were adjusted. Dunaliella tertiolecna and yeast are used as food. Feeding was based on water color. They were maintained for 25 days under the notified circumstances. On days 5, 10, 15, 20 and 25, Survival and growth rates in all treatments were calculated by counting the number of existent Artemia versus the whole Artemia in the first day.


Survival and growth rate during the experiment showed that with salinity increasing, Artemia viability decreases. In salinity 45 ppt, the lowest survival rate was observed and in the salinity of 40 ppt, higher survival and growth rate were observed. On the fifth day, after the calculations have been made, the lowest survival and growth where observed in salinity 45 ppt and the highest were observed in 35 ppt. At the end of day 25, the highest growth and survival rate was observed in salinity 40 ppt and the lowest growth rate was perceived in salinity 45 ppt, as the beginning of the experiment.


In the present study, the effect of salinity on Gavkhoony Artemia growth and survival was calculated. The lowest growth and survival rate were observed in 45 ppt salinity. That's because in the high salinity water, the food was demolished and it become out of reach. They have been used Dunaliella tertiolecna as a food very well in all the thanks

except in 45 ppt tank. In 45 ppt thank, salt were deposited on the bottom and the algae were destroyed and the food was out of reach of Artemia. Total length decreasese in high salinity. The biometery datas showed that total length in the tenth day decreases according salinity increases. An increase in the salinity has a negative effect on the growth rate. These results confirm the prior data. Results have shown that Growth rate has an inverse relationship to salinity.

Hoopes Browne (1990) was found a high mortality in the Artemia culture tank with 150ppt salinity. Regarding to their report, before second day of treatment the whole Artemia where dead in the tanks that had 230 to 250 ppt salinity. In this study, high salinity (>150 ppt) was harmful for Artemia growth and survival (6). Lotfi (2002), poved that among all the effective factors, salinity is the less effective factor in the growth rate. Based on the results, Gavkhuni Artemia survival decreases in high salinity water (7).


Sorgeloos, P., Dhert, P., Candreva, P., 2001. Use of brine shrimp Artemia spp., in marine fish larviculture, Aquaculture, 200, 147-159.

Van Stappen, G., 1997. Introduction, Biology and ecology of Artemia, Manual on the production and use of live food for aquaculture, FAO fisheries technical paper 361, Rome, FAO, PP.101-150.

Grailou, Z., 2001. Stability of long-chain polyunsaturated fatty acids in Artemia enriched with various oils and periods of starvation, Fisheries Master's Thesis, AlphaDepartment of Natural Resources, Tehran University, pp. 224.

Agh, n., 1997, Effect of physico-chemical factors on the hatching of the Artemia urmiana cysts. First conferences of zoology in Iran, Tarbiat Moallem University, Tehran, Iran.

Kolkovski, S., Curnow, J., King, J., 2004. Intensive rearing system for fish larvae research II Artemia hatching and enriching system. Aquaculture Engineering, 31, 309-317.

Browne, R.A., C.W Hoops., 1930. Genotype diversity and selection in asexual brine shrimp (Artemia). Evolution, 44, 1035-1051.

Lotfi, V., Agh, N., Sepehri, H., 2002. Effect of various salinity levels on survival, growth rate, reproductive characteristics and longevity of three Iranian Artemia populations. Artemia and aquatic animal investigation institute, Urmia, Iran.

Organisms - inhabitants of the Crimean salt lakes as live food in larviculture

N.V. Shadrin, E.V. Anufriieva

Institute of Biology of the Southern Seas, Sevastopol, Ukraine; e-mail: snickolai @ yandex.ru

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