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Page 1: ksat 201903 1 ctp - Khazar Journal of Science and ... · KHAZAR JOURNAL OF SCIENCE AND TECHNOLOGY (KJSAT) Hamlet Isayev/Isaxanli * Editor-in-Chief Khazar University, Azerbaijan Editorial
Page 2: ksat 201903 1 ctp - Khazar Journal of Science and ... · KHAZAR JOURNAL OF SCIENCE AND TECHNOLOGY (KJSAT) Hamlet Isayev/Isaxanli * Editor-in-Chief Khazar University, Azerbaijan Editorial

Vol. , № , 201 ISSN 2520-6133

KHAZAR JOURNAL OF

SCIENCE AND TECHNOLOGY (KJSAT)

Hamlet Isayev/Isaxanli * Editor-in-Chief

Khazar University, Azerbaijan

Editorial Board

Abdul Latif Bin Ahmad Universit Sains

Malaysia

Andrey Kuznetsov North Carolina State University,

USA

Yuri Bazilevs University of California, San Diego,

USA

Jinhu Lü Academy of Mathematics and Systems

Science, Chinese Academy of Sciences, China

Chunhai Fan Shanghai Institute of Applied Physics, Chinese

Academy of Sciences, China

Shaher Momani Mutah University,

Jordan

Davood Domiri Ganji Babol Noshirvani University of Technology,

Iran

Bernd Nilius KU Leuven,

Belgium

Mehrorang Ghaedi Yasouj University,

Iran

Asaf Salamov Lawrence Berkeley National Laboratory,

USA

Madjid Eshaghi Gordji Semnan University,

Iran

Kenji Takizawa Waseda University,

Japan

Tasawar Hayat Quaid-I-Azam University, Islamabad,

Pakistan

Tayfun Tezduyar Rice University,

USA

Lei Jiang Institute of Chemistry, Chinese Academy of

Sciences, China

T. Nejat Veziroglu University of Miami,

USA

*Also H. Issakhanly, H. Isakhanly, H. Isakhanli, H.A.Isayev, G.A.Isayev, or G.A.Isaev due to differences intransliteration. I use Hamlet Isayev in mathematics and science fields and Hamlet Isakhanli/Isaxanli in humanities.

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Copyright © Khazar University Press 201All Rights Reserved

Managing EditorsKhazar University, Azerbaijan

Nuru SafarovIrada Khalilova Mahammad Nuriyev Rovshan Abbasov

Ali HuseynliNazimRasoul MoradiJavid jaghi

Khazar University41 Mehseti str., Baku AZ1096

Republic of AzerbaijanTel: (99412) 4217927

Website: www.kjsat.com

KHAZAR UNIVERSITY PRESS

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Contents

Characterization of Azerbaijan Durum Wheat Accessions Statistical Analysis ..............................................................................................5

Asaf Omarov, Moozhan Serpoush, Hawa Adli, Irada Khalilova Detection of Antibiotic Resistance Bacterial Isolates from Retail Chickens and Eggs in Azerbaijan ............................................................................................................................14

Moozhan Serpoush, Farzad Afshari, Manoochehr Khodarahmi, Javid Ojaghi Race Analysis of Puccinia Striiformis f.sp. Tritici in Iran. ..................................................27

Allahverdi ShahveranovDo CRISPR or CRISPR-like Systems and Sequences Exist in Human Mitochondria or Evolutionary Disappeared in Human Cells? ......................................................................37

Rustam Rustamov Conceptual Approach of Regional Problem of the Global Climate Change........................39

Ulduza Gurbanova, Shahniyar Bayramov, Irada Huseynova The Dynamics of Some Carbon and Nitrogen Metabolism Enzymes in Daytime inVarious Wheat Genotypes during Drought..........................................................................46

Kubra Yusifova Sensitivity of Primary Tripsinized Cell Systems EYQ and FEC to the Fowl Pox Virus .......53

Lale Aydinli, Durna Aliyeva Diurnal Temperature-Related Dynamics of Glutathione Reductase Activity in Wheat Genotypes Under Drought ...................................................................................................58

Arailym Nurmasheva Knowledge, Attitudes and Perceptions (KAP) on Point of Sale (PoS) Advertising and Promotion of Tobacco Bans Among Parents: A Cross-Sectional Study in Nur-Sultan, Kazakhstan...............................................................................................................64

Information to Contributors .............................................................................................84

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Khazar Journal of Science and Technology Volume 3 №1 2019, 5-13 © Khazar University Press 2019 DOI: 10.5782/2520-6133.2019.3.1.5

5

Agro-morphological Characterization of Azerbaijan Durum Wheat Accessions by Multiple Statistical

Analysis

Gamar Safarova1, Ellada Akhundova1, Maryam Ramazanova1, Samira Salayeva1, Javid Ojaghi2*

1Department of Genetics and Theory of Evolution, Baku State University, Baku, Azerbaijan

2Life Sciences department, Khazar University, Baku Azerbaijan *corresponding author: [email protected]

Abstract

The present investigation was conducted, using biometrical analysis, to estimate the genetic diversity of biomorphological traits in 69 durum wheat accessions. The traits evaluated were plant height, number of fertile tillers, spike length, number of spikelets, number of grains per spike, grain weight per spike, grain yield and 1000 kernel weight. Analysis of variance demonstrated significant differences between durum wheat samples for the all the traits studied. Coefficient variation indicated that grain yield, spike length and grain weight per spike resulted from high genetic diversity between the traits studied. Correlation analysis showed that genotypes with high yield, had high numbers of grains per spike, high grain weight per spike and high 1000 kernel weight. Cluster analysis classified genotypes into seven main groups, so that genotypes with different characteristics were located in separate groups. Results showed that there was adequate genetic variability in Azerbaijan durum wheat germplasm to support breeding for improved grain yield and that indirect selection for high yield can be conducted in early generations using traits that include number of grains per spike, grain weight per spike and 1000 kernel weight.

Keywords: Durum wheat, genetic diversity, multiple statistical, morphological traits

Introduction

Durum wheat (Triticum turgidum L. var. durum) is the only tetraploid species of wheat of commercial importance in wide cultivation today (Shewry, 2009). Despite

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6 Safarova et al.

its smaller growing area and lower annual production than hexaploid wheat, durum wheat has long remained an important food resource for the human diet. It is used in different parts of the world for a range of food products, such as pasta, couscous, unleavened bread, bulgur, and mote, among others (Nachit, 1992). Turkey and Canada are the countries producing most, each with an estimated 2 million ha (USDA, 2015), followed by Algeria, Italy and India, each cultivating over 1.5 million ha (Bonjean et al., 2016). Syria belonged to this group of large producers, but the recent unrest has greatly reduced crop production. France, Greece, Morocco, Pakistan, Portugal, Kazakhstan, Russia, Spain and Tunisia cultivate durum wheat on areas from 0.5 to 0.8 million ha annually (USDA, 2015). Azerbaijan, Iraq and Iran combined grow durum wheat on over 0.7 million ha (Bonjean et al., 2016).

Information on genetic diversity and the relationships among and between individuals, populations, plant varieties, animal breeds and species is important to plant and animal breeders for the improvement of crop plants and animal breeds, for conservation biology and for studies of the evolutionary ecology of populations. It is essentially the first step towards plant breeding for crop improvement and is immediately available from germplasm, regarded as a reservoir of variability for different characteristics (Wondifraw, 2016).

Morphological markers are based on visually accessible traits such as flower colour, seed shape, growth habits and pigmentation; this does not require expensive technology, but does often require large tracts of land for field experiments. These marker traits are often susceptible to phenotypic plasticity; conversely, this allows assessments of diversity in the presence of environmental variation, which cannot be neglected in determining genotypic variation. These types of markers are still advantageous and are indeed mandatory in distinguishing adult plants from genetic contaminations in the field, for example, spiny seeds, bristled panicle and variants in flower and leaf colouration (Govindaraj et al., 2015).

As for the gene bank of Azerbaijan, however, little has been done to characterize durum wheat accessions based on studies of a large number of qualitative and quantitative traits and the interrelationships among those traits with greatest effect on genotype discrimination. More information, however, is needed to identify adapted accessions suitable for a breeding programme for durum wheat in Azerbaijan. Therefore, the main objectives of this investigation were, firstly, to evaluate the morphological traits of 69 durum wheat accessions for use in describing the genetic variation of accessions from several countries preserved in Azerbaijan’s national gene bank and, secondly, to study the relationships between the traits studied using multiple statistical analysis.

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Agro-morphological Characterization of Azerbaijan Durum Wheat Accessions by Multiple Statistical Analysis 7

Materials and methods

In this study, we used durum wheat (Triticum durum L.), 69 accessions by base collection of the Azerbaijan National Gene Bank (Table 1). The study was conducted at the Genetic Absheron Experimental Station of Genetic Resources Institute of the ANAS. Experiments were conducted on the basis of RCBD experimental design with three replications during the autumn season of 2018-2019. Each accession was planted in a 1m long plot with a between-row spacing of 25 cm and within-row spacing of 10 cm. Observations were recorded on 10 randomly selected competitive plants for eight morphophysiological traits: plant height (cm), Number of fertile tillers, spike length (cm), number of spikelets per spike, number of grains per spike, grain weight per spike (gr), grain yield per plot (g) and 1000 kernel weight. SPSS and PAST programmes were used for statistical analyses of correlation, principal component analysis and genotype clustering.

Table 1. Names of variety and pedigree of the durum wheat used in this study No.

Variety name Pedigree No.

Variety name Pedigree

1 Mut.leucurum tur.luzit x leucomelan

36 Erythromelan murciense x leucomelan

2 Murciense erythroleuc x murciense

37 Leucomelan murciense x leucomelan

3 Murciense tur.luzit x miltur 38 Apulicum melanopus x murciense

4 Murciense lut x murciense 39 Hordeiforme melanpus x murciense

5 Affine Bezostaya 1 x leucur

40 Leucomelan leucurum x mut.apulicum

6 Mut.murciense graecum x aegyption

41 Hordeiforme melanopus x murciense

7 Obscurum lut(Azəri x niloticum)

42 Mut. Murciense leucurum x mut. apulicum

8 Obscurum graecum x niloticum

43 Mut.alexsandrinum

leucurum x murciense

9 Hordeiforme Azəri x Sevinc 44 Melanopus melanopus x hordeiforme

10 Melanopus tur.albojadur x hostion

45 Hordeiforme mut.hordeiforme x leucurum

11 Africanum albidium x lybic 46 Lybicum boeuffix x leucurum

12 Melanopus turgid

Bezostaya 1 x erythromelan

47 Coerulescens mut.lybicum x leucurum

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13 Mut.lybicum albidium x lybicum

48 Erythromelan mut.hordeiforme x leucurum

14 Mut.lybicum coerules x polnic 49 Hordeiforme apulicum x graecum

15 Niloticum Abşeron 50 Reinchebachii apulicum x graecum

16 Obscurum lutescens x obscurum

51 Mut.apulicum mut.apulicum x graecum

17 Obscurum hostianum x obscurum

52 Mut.lybicum lybicum x lutescens

18 Obscurum hostianum x erythrospermum

53 Etyhromelan hordeiforme x leucomelan

19 Albooscurum Bezostaya 1 x lybicum

54 Leucurum niloticum x leucurum

20 Albooscurum erythrospermum x obscurum

55 Mut.hordeiforme lutescens x sarı buğda

21 Alboprivinciale Abşeron 56 Mut.hordeiforme lutescens x erythromelan

22 Alboprivinciale Bezostaya 1 x lybicum

57 Niloticum erythrospermum x apulicum

23 Mut.africanum albidum x lybicum

58 Mut.hordeiforme hordeiforme x lutescens

24 Aegiptiacum Bezostaya 1 x niloticum

59 Affine affine x eryhromelan

25 Lubicum Azəri x coerulescens

60 Hordeiforme sarı buğda x lutescens

26 Hordeiforme apulicum x hordeiforme

61 Affine şərq x miltirum

27 Affine leucomelan x affine

62 Mut.murciense sevinc x velutinum

28 Hordeifrme Azəri x erythromelan

63 Murciense sevinc x velutinum

29 Murciense Bezostaya 1 x lybicum

64 Aegiptiacum sevinc x velutinum

30 Affine murciense x leucurum

65 Murciense sarı buğda x gürgənə

31 Murciense murciense x leucurum

66 Hordeiforme hordeiforme x erythrospermum

32 Leucomelan apulicum x leucomelan

67 Murciense hordeiforme x erythrospermum

33 Niloticum apulicum x murciense

68 Leucurum leucurum x milturum

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Agro-morphological Characterization of Azerbaijan Durum Wheat Accessions by Multiple Statistical Analysis 9

34 Murciense apulicum x murciense

69 Hordeiforme leucurum x milturum

35 Melanopus leucomelan x melanopus

Results and discussion

The descriptive statistics for the traits of the wheat genotypes evaluated are listed in Table 2. The coefficient of variation for most of the traits displayed high diversity among the genotypes studied. The high coefficient variation in most of the traits studied is very useful for germplasm improvement. In this study the highest CV was observed in grain yield, at 61.52 %. The traits’ spike length, grain weight per spike, number of grains per spike and 1000 kernel weights also displayed high genetic diversity. The high variation in plant height indicates that breeders can use cultivars of appropriate height in breeding programmes for mechanized harvesting and to counter lodging risk. Similar results were obtained by Babaie Zarch et al. (2013). The high genetic diversity of the traits studied in our research indicates that selection for increased value may be highly effective.

Table 2. Statistical indicators of traits in the durum wheat accessions studied Traits Min. Max. Mean Standard

error of mean

Standard deviation

Coefficient variation (CV%)

Plant height 75.30 173 130.387 2.759 22.916 17.575 Number of fertile

tillers 3.20 4.20 3.631 0.03 0.253 6.97

Spike length 5.5 27.80 8.699 0.329 2.734 31.43 Number of spikelets

16 35.20 24.278 0.395 3.289 13.55

Number of grains per spike

16.40 70.20 47.971 1.221 10.149 21.16

Grain weight per spike

1.0 3.98 2.235 0.069 0.57 25.5

Yield 1.0 13.52 2.373 0.176 1.46 61.52 1000 kernel

weight 30.04 76.10 47.191 1.112 9.236 19.57

Spike length and number of grains per spike are important to grain yield. Genetic variance was observed at 7.13. Arya et al. (2018), Yadav et al. (2006), Nagireddy and Jyothula (2009) and Kaul and Singh (2011) demonstrated high heritability with high genetic advance for these traits. This indicates a substantial contribution of

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10 Safarova et al.

additive genetic variance in the expression of these characters and may be useful in hybridization and selection for higher grain yield.

Selection programmes to increase 1000 kernel weight as a principal component of grain yield may play an important role in improving grain yield. This research indicated high genetic diversity for this trait. Analysis of variance demonstrated a significant difference between the wheat genotypes for all traits studied. The results of correlation (Table 3) between the traits measured demonstrated a significant positive relationship between plant height, number of spikelets per spike, number of grains per spike and grain weight per spike.

Significant positive correlation was recorded between grain yield, number of grains per spike, grain weight per spike and 1000 kernel weight. Hence, these traits may be considered key yield components in selection processes for the breeding of durum genotypes for high yield.

The cluster analysis based on Ward’s method classify all the durum wheat accessions studied in 7 main groups (Fig. 1). The genotypes No.37 and 65 were grouped into the first cluster, which had the lowest mean for plant height and the highest 1000 kernel weight. This group also shows that in plants of low height, this reduced height increases 1000 kernel weight, one of the traits affecting yield. The cultivars in this group are suitable for breeding programme to create plant varieties of low height.

11 genotypes were clustered into the second group, consisting of genotypes with the greatest spike length of the genotypes studied. Cluster III consisted of 4 cultivated types: No. 12, 41, 44 and 45, principally characterized by high traits in plant height, grain weight per spike and grain yield. These genotypes are strongly recommended as high yield parents for use in future selection programmes. Cluster IV, comprising 15 durum wheat samples, demonstrated high grain weight per spike. In total, 8 samples were grouped in cluster V and 12 wheat samples in cluster VI. Finally, cluster VII contained 17 genotypes, comprising 24.63% of all the genotypes examined. Conducting multivariate analysis, Singh et al. (2018) studied 35 genotypes, which were grouped into 6 clusters based on genetic divergence value.

The results of this research introduced information about the genetic structure of durum wheat accessions in Azerbaijan which should be considered in the further collection of genetic resources and which demonstrate a wide spectrum of agronomic variability in the genotypes investigated.

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Agro-morphological Characterization of Azerbaijan Durum Wheat Accessions by Multiple Statistical Analysis 11

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Agro-morphological Characterization of Azerbaijan Durum Wheat Accessions by Multiple Statistical Analysis 13

References Bonjean, A. P., Angus, W. J., & Ginkel, M. (2016). The World Wheat Book: A History of

Wheat Breeding; Lavoisier: Paris, France, Volume 3. Govindaraj, M., Vetriventhan, M., & Srinivasan, M. (2015). The Importance of Genetic

Diversity Assessment in Crop Plants and Its Recent Advances: An Overview of Its Analytical Perspectives. Genetics Research International, Article ID 431487, 14 pages.

Kaul, D. K., & Singh, B. (2011). The Evolution of Drought Tolerance in Elite Genotypes of Bread Wheat (Triticum aestivum L.). Advances in Plant Sciences, 24(1), 141-144.

Babaie Zarch, M. J., Fotokian M. H., & Mahmoodi, S. (2013). An Evaluation of Genetic Diversity in Wheat (Triticum aestivum L.) Genotypes for morphological traits using multivariate analysis methods. Journal of Crop Breeding Vol. 5, No. 12.

Nachit, M. M. (1992). Durum wheat breeding for the Mediterranean drylands of North Africa and West Asia. In: Rajaram, S., Saari, E.E., Hettel, G.P. (eds.). Durum Wheats: Challenges and Opportunities. CIMMYT. Ciudad Obregon, Mexico. p.14-27.

Nagireddy, A. V., & Jyothula, D. P. B. (2009). The Heritability and interrelationship of yield and certain agronomic traits in wheat. Res. on Crops, 10(1), 124-127.

Wondifraw, M. S. (2016). The genetic diversity and selection efficiency of Ethiopian durum wheat (Triticum durum Desf.) landrace collections. Degree of Master of Science (M.Sc.) in biology.

Shewry, P. (2009). Increasing the health benefits of wheat. FEBS Journal 276:71-71. Singh, G., Kumar, P., Kumar, R., & Gangwar, L. K. (2018). Genetic diversity analysis of

various morphological and quality traits in bread wheat (Triticum aestivum L.). Journal of Applied and Natural Science 10 (1): 24 - 29.

USDA Foreign Agricultural Service. (2015). Grain and Feed Annual; GAIN Report No: TR5016; USDA Foreign Agricultural Service: Ankara, Turkey.

Arya, K., Singh, J., Kumar, L., Singh Nagar, S., Ahalawat, N. K., & Chand, P. (2018). Studies of genetic variability and heritability in relation to grain yield and its component traits in wheat (Triticum aestivum l.). Int. J. Agricult. Stat. Sci. Vol. 14, No. 1, pp. 215-222.

Yadav, D. K., Pawar, I. S., Sharma, G. R., & Lamba, R. A. S. (2006). Genetic diversity in some germplasm lines of bread wheat. J. of Plant Improvement, 8(2), 138-141.

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Khazar Journal of Science and Technology Volume 3 №1 2019, 14-26 © Khazar University Press 2019 DOI: 10.5782/2520-6133.2019.3.1.14

14

Detection of Antibiotic Resistance Bacterial Isolates from Retail Chickens and Eggs in Azerbaijan

Asaf Omarov*, Moozhan Serpoush,

Hawa Adli, Irada Khalilova

Department of Life Sciences, Khazar University, Azerbaijan *Corresponding author: [email protected]

Abstract

Microbial Resistance to antibiotics is on the rise, in part because of inappropriate use of antibiotics in human medicine but also because of practices in the agricultural industry. Intensive animal production involves giving chicken large quantities of antibiotics to promote growth and prevent infection. These uses promote the selection of antibiotic resistance in bacterial populations. The resistant bacteria from agricultural environments may be transmitted to humans, in whom they cause disease that cannot be treated by conventional antibiotics. The objective of this study was to investigate the antibiotic resistance of bacteria isolated from commercial broiler chicken farms and village chicken farms.

Keywords: Antibiotics, resistance, animal products

Introduction

In modern food animal production, antimicrobial agents are used in one of four different ways. Therapy: treatment of infections in clinically sick animals, preferably with a bacteriological diagnosis. Metaphylactics: treatment of clinically healthy animals belonging to the same flock or pen as animals with clinical symptoms. In this way infections may be treated before they become clinically visible and the entire treatment period thereby shortened (Aarestrup, 2005; Hao; Cheng et al, 2014). In modern productions systems this is the only way to treat large broiler flocks with water medication. Prophylactics: treatment of healthy animals to prevent disease in a period where they are stressed (e.g. medicated early weaning) (Munita and Arias, 2016). This use of antimicrobial agents may indicate management problems, and is in most countries not legal or considered imprudent. Growth promotion: continuous inclusion of antimicrobial agents in animal feed to improve growth (Blair et al,

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Detection of Antibiotic Resistance Bacterial Isolates from Retail Chickens and Eggs in Azerbaijan 15

2015). Antimicrobial agents are medicines used to treat infections caused by bacteria in particular (Nathan and Cars, 2014; Lushniak, 2014). They are essential to both human and animal health, but in recent years, some bacteria have demonstrated full or partial resistance to various antimicrobial agents (Aslam et.al, 2018; Lye et al, 2012; Molbak, 2004). This phenomenon, called antimicrobial resistance (AMR), is rising concern for both public and animal health (Nathan and Cars, 2014; Bonnie and Marshall, 2011). Many of the actions implemented to improve animal health depend on the availability and appropriate use of quality veterinary medicines, and notably antimicrobial agents (Nathan and Cars, 2014). Animal health is a key component of policies to improve animal welfare, food security and food safety (ISO 6887; Littmann and Viens, 2015).The OIE believes it is vital to enable adequate access to effective antimicrobial agents to treat animal diseases, but emphasises the need to regulate that access through the intervention of well-trained veterinarians, whose ethics are ensured by national Veterinary Statutory Bodies as laid down by law (Nathan and Cars, 2014). Veterinary practice Antibiotics not only administered prophylactically but subtherapeutic doses are administered routinely via feed to increase feeding efficiency such as rate of weight gain in poultry subtherapeutic doses of antibiotics in feed promotes bacterial resistance by decimating the susceptible population of micro-organisms normally present in animals, selecting drug resistant strains (Cavalieri et al, 2005; O’Neill, 2016).

If drug-resistant bacteria spread to humans through the consumption of animal products, resistant bacteria could colonize their new hosts transferring antibiotic resistance to the normal drug-susceptible bacteria already present in the human gut flora (Blair et al, 2015; Randall et al, 2013).

The primary aim of this study was to estimate the proportion of isolates resistant to specified antimicrobials amongst E. coli, Salmonella spp., Enterococcus spp. isolated from the gut of Azerbaijan meat chickens.

Materials and Method

The samples were collected between August 2018 and January 2019. The methods followed for this study are in line with recommendations from the OIE Chapter 6.7 “Harmonisation of national antimicrobial resistance surveillance and monitoring programmes” (OIE Standards. 2015; OIE. 2018; Veldman et al, 2017) and ISO 11133:2014 Microbiology of food, animal feed and water – Preparation, production, storage and performance testing of culture media (ISO, 2014; ISO, 2017; Singer et al, 2016). Chicken samples and eggs were collected from retail markets and the bacterial cultural were obtained from the chicken samples and eggs (ISO, 2018; ISO/TS 17728; Sohail et al, 2016).

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16 Omarov et al.

Whole chickens were putted sterile plastic bags and added 1000ml sterile isotonic solution and washed, shaken 15 minutes. 1ml suspension was taken and resuspend the particles (1:10-1:1000) and each dilution (100mikrolitr) streaked direct onto SBA agar. The agar plates were incubated at 37°C for 24 h. CFUs were counted simple tick methods and multiply the dilution quantity and founded average numbers of CFUs (ISO 17604; Singer et al, 2016).

The eggs were putted sterile plastic bags and added 100ml sterile isotonic solution and washed, shaken 15 minutes. 1ml suspension was taken and resuspend the particles (1:10-1:1000) and each dilution(100mikrolitr) streaked direct onto SBA agar. The agar plates were incubated at 37°C for 24 h. CFUs were counted simple tick methods and multiply the dilution quantity and founded average numbers of CFUs (Holmes et al, 2016; ISO, 17604; Nathan and Cars, 2014).

100mikl yolk and white from eggs were suspended sterile isotonic solution different tubes and resuspend the particles (1:10-1:1000) and each dilution(100mikrolitr) streaked direct onto SBA agars. CFUs were counted simple tick methods and multiply the dilution quantities and founded average numbers of CFUs (ISO 6579-1:2017; ISO 6887; ISO 6887-1:2017; ISO 7218).

Enterococcus isolation and typing

The prepared sample was shaken to resuspend the particles, and then streaked direct onto SBA agar. The agar plates were incubated at 37°C for 24 h. From a pure subculture from the original colony, bacteria were harvested for storage at -20°C on cryo-beads in two separate, identical containers labelled with the sample code and the laboratory reference number (ISO 6887-1:2017; Lynch et al, 2013; Murray, 1990).

E.coli isolation and typing

E.coli was isolated using the ISO 16649-1:2018 for E.coli spp. using SBA, Endo agar and Bismuth sulfite agar and incubated at 37°C for 24h (Dodani, 2018; ISO 16649-1:2018; Islam et al, 2016).

The prepared sample was shaken to resuspend the particles, and then streaked direct from SBA onto Endo agar which achieved both bacterial isolation and type confirmation. The agar plates were incubated at 37°C for 18h and then one clone was selected and subcultured onto Bismuth sulfite agar. E. coli isolation was confirmed using an indole test. From a pure subculture from the original colony, bacteria were harvested for storage at -20°C on cryo-beads in two separate, identical

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Detection of Antibiotic Resistance Bacterial Isolates from Retail Chickens and Eggs in Azerbaijan 17

containers labelled with the sample code and the laboratory reference number (ISO 16649-1:2018; Kalia et al, 2014).

Salmonella spp. isolation and typing

Salmonella was isolated using the method ISO 6579:2002 for Salmonella spp. using SBA, Endo agar and Bismuth sulfite agar and incubated at 37°C for 24h. Bacterial isolates were harvested for storage at -20°C on cryo-beads in two separate, identical containers labelled with the sample code and the laboratory reference number (ISO 6579-1:2017;Wellington et al, 2013).

Each isolated and identified colonies have been submitted to an antibiogram test carried out by the disk diffusion method, as recommended by the National Committee for Clinical Laboratory Standards (Bonnie and Marshall, 2011).

Antimicrobial susceptibility for the isolates was determined by the broth microdilution method either on veterinary reference card panels according to the manufacturers’ guidelines or in-house panels prepared according to Clinical and Laboratory Standards Institute (CLSI) standards. Isolates were subjected to analysis using both Clinical Breakpoints and Epidemiological Cut-off Values (ECOFF) (Liu et al, 2010).

Isolates were screened for the following antibiotics and doses: ceftriaxone (CFX) 30 mg, gentamicin (GEN) 10 mg, tetracycline (TET) 30 mg, erythromycin (ERI) 15 mg, amoxicillin (AX) 25 mg. More this concentration considered as antibiotic resistant bacteria (Bonnie and Marshall, 2011; Singer et al, 2016; Smith et al, 2007; Singer et al, 2016).

Results

Different antimicrobial resistance patterns were identified from all chicken samples. 8 bacterial strains (4 Enterococcus spp., 2 E.coli, 2 Salmonella spp.) were isolated from the chicken of the different poultry farms (Table 1). Total bacterial counts (colony forming units-CFU) were underestimation which was given ISO standards. SB, HG, SN, VC conventional names were given to poultry farms. Enterococcus spp. was detected from SB, HG, SN and VC, E.coli was identified from SN and VC, Salmonella spp. was sensed HG and VC. Enterococcus spp., E.coli. and Salmonella spp. were isolated in VC.

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18 Omarov et al.

Table 1. Isolated bacterial strains from the chicken of the different poultry farms

Enterococcus spp. E.coli Salmonella spp.

SB + - -

Hg + - +

Sn + + -

Vc + + +

In order to determine resistances, all strains were tested susceptibility to 5 antibiotics. The results are summarized in Table 2.

The isolates screened, the most common resistance was observed against gentamicin, amoxicillin and erythromycin. Although 62.5% isolates were resistance to ceftriaxone and 62.5% isolates were resistance to tetracycline. Enterococcus spp (SN), E.coli (SN), Enterococcus spp.(VC), E.coli(VC) were perform resistance to ceftriaxone but Enterococcus spp (SB) and both Salmonella spp. strains were perform sensitivity to ceftriaxone. Enterococcus spp (SB), Enterococcus spp(HG), Salmonella spp.(HG), Salmonella spp.(VC) were perform resistance but Enterococcus spp (SN), E.coli (SN), E.coli (VC) were perform sensitivity to tetracycline. Salmonella spp (VC) resistance is increasing against ceftriaxone (Table 2).

Enterococcus spp. achieve the most common resistance to antibiotics. Salmonella spp (VC) perform resistance against gentamicin, tetracycline, amoxicillin and erythromycin but both isolates of Salmonella spp perform the sensitivity against ceftriaxone. E.coli spread resistance to gentamicin, amoxicillin and erythromycin but thoughtful to tetracycline (Table 3).

Discussion The results show that Enterococcus spp are commonly found in the poultry farms but CFU level is underestimation. Antibiotic resistance Enterococcus spp were isolated from broiler chickens in terms of distribution among farms. Our results showed that Enterococcus spp strains were resistant to amoxicillin, gentamicin, erythromycin. Though the enterococci in this study showed acquired resistance traits to a number of antibiotics, they did generally not show resistance to the clinically relevant antibiotic tetracycline and ceftriaxone, especially among the Enterococcus spp (HG), Enterococcus spp (SB) strains. Most challenging are strains that have acquired multiple antibiotic resistance, especially resistance to erythromycin, amoxicillin and gentamicin. It is difficult to assess the impact of antibiotic-resistant

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Detection of Antibiotic Resistance Bacterial Isolates from Retail Chickens and Eggs in Azerbaijan 19

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Detection of Antibiotic Resistance Bacterial Isolates from Retail Chickens and Eggs in Azerbaijan 21

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22 Omarov et al.

enterococci from foods on potential human pathogenicity. For the reason that antibiotic resistance alone cannot explain the virulence of enterococci. In order to become pathogenic, they need to express virulence traits associated with adhesion, translocation, and evasion of immune responses and cause pathological changes. It is clear that in the hospital environment, antibiotics may influence selection of pathogenic enterococci, which may lead to infections or superinfections (Molbak, 2004). Eaton and Gasson showed that the incidence of virulence factors was highest among clinical enterococcal isolates, followed in decreasing order by food strains and starter strains, suggesting that the food and starter strains have a lower potential for pathogenicity (CLSI, 2012).

E.coli and Salmonella spp isolates were recovered from VC and HG poultry farms. Obtaining such bacteria of the chicken samples from retail markets were due to the bad safety practice, which followed poultry farms. Similar antibiotic resistance patterns were observed in different farms, indicating that certain isolates were clonal. Clonal isolates could also play an important role in the emergence of antibiotic-resistant Escherichia coli strains. Forgetta and et al came same conclusion for Escherichia fergusonii (Davies and Davies, 2010) High levels of resistance to gentamicin, amoxicillin and erythromycin could be due to extensive use of this antibiotics over the years since its introduction to feed animals. This finding differed from that of Rooklidge (Pena-Miller et al, 2013; Rooklidge, 2004). The observation in the present study of isolates resistant to amoxicillin is in contrast with that reported by Smith et al. (Viktória et al, 2018; Smith, 2007) in E. coli isolated from poultry. Co-resistances were observed in the vast majority of strains this is due to co-selection with other resistance factors.

Two Salmonella isolates displayed resistance to the gentamicin, tetracycline, amoxicillin, erythromycin, but both strains of salmonella were susceptible to ceftriaxone. Of particular importance is the isolation of tetracycline and erythromycin resistant salmonella. The latter can cause severe illness. The ability of bacteria to acquire antibiotic-resistance genes and subsequently spread them to many different bacterial species is well known (Hall, 1997).

Our finding of the two salmonella isolates and E.coli from two brands of chicken samples from two grocery stores demonstrates the potential for the contamination of food during handling and processing. However, the contamination of retail meats with resistant salmonella mainly reflects carriage of the organism by poultry farms; intervention strategies should therefore focus principally on reducing the number of pathogens present on farms and in slaughterhouses.

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Detection of Antibiotic Resistance Bacterial Isolates from Retail Chickens and Eggs in Azerbaijan 23

Variations in husbandry practices among farms may play a role in the antibiotic resistance profile of isolates, because a significant difference was noticed between different farms.

Conclusions

Antibiotic resistance is one of the most serious threats to human health today (World Health Organization.2014). We should seek to increase our knowledge regarding the extent of the AMR issue. Ecological and environmental aspects of the issue need not be ignored; all the elements of “one health” should be part of the control policy. Alternative strategies may also play a fruitful role, especially in developing countries. Current global interest indicates that AMR is not an unheeded issue anymore. Although this attention is not itself adequate to combat AMR, a global code of conducts implementing all the options of action against AMR might eliminate in the future (Roca et al, 2015). The creation of new antibiotics targeting the growing threat of multidrug resistance is a goal that remains "alarmingly elusive” (Merelli et al, 2013). Alternatives to antibiotics such as probiotics and lytic bacteriophages can help to decrease the burden of AMR globally. The threat of resistance will always accompany any new drug introduced for clinical use. The only possible, sustainable solution is to keep pace with it. This will involve introducing profound changes in the use of these drugs, including stewardship programs for rational use and improve targeted therapy. Furthermore, implementation of adequate preventive measures such as vaccines and faster diagnostic tools as well as improving hygiene and reducing the use of antibiotics in animals, will be the only way for preserving the usefulness of antibiotics for future generations and ensure a healthy future for the world’s population. Finally, it is of critical importance to acquire a more comprehensive understanding of the molecular, evolutionary and ecological mechanisms governing the spread of antibiotic resistance. It is time to distinguish the exact costs of antibiotic use in agricultural practice in terms of antibiotic resistance and its significances on the sustainability of susceptible bacterial flora in the environment and to act accordingly.

Acknowledgements

The authors acknowledge research funding from Khazar University

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Khazar Journal of Science and Technology Volume 3 №1 2019, 27-37 © Khazar University Press 2019 DOI: 10.5782/2520-6133.2019.3.1.27

27

Race Analysis of Puccinia Striiformis f.sp. Tritici in Iran

Moozhan Serpoush*1,2, Farzad Afshari 3, Manoochehr

Khodarahmi 3, Javid Ojaghi2

1Department of Plant Breeding, Science and Research Branch, Islamic Azad University, Tehran, Iran.

2Department of Life Sciences, Khazar University, Baku, Azerbaijan 3 Seed and plant Improvement Institute, Agricultural Research Education

and Extension Organization (AREEO), Karaj, Iran. *Corresponding author: [email protected]

Abstract

Globally, wheat is an important crop; as a strategic plant it occupies the largest area of cultivation. Puccinia striiformis f.sp. tritici, known as yellow rust, caused the embrace damage and epidemic in Iran. Resistance cultivars are known to be the best way to control and prevent the spread of rust. This research studied the genetics of the pathogenicity of 26 isolates of stripe rust from different important wheat-growing areas in Iran; 56 differential and isogenic lines were used with a Bolani susceptible check, under greenhouse condition. Race 6E6A+,Yr27 from Neishabour (Eshgh Abad) and 7E22A+,Yr27 from Kermanshah were found to be less aggressive races in this research and races 206E182A+,Yr27 from Islam Abad, 207E190A+,Yr27 from Fars and race 231E150A+,Yr27 from Mashhad, with more than 19 known wheat genes, were the most aggressive races. Results of this research were that virulence was observed on plants with genes Yr1, Yr2, Yr3, Yr6, Yr7, Yr8, Yr9, Yr18, Yr21, Yr25, Yr26, Yr28, Yr29, Yr31, Yr32, YrSU, YrND, YrCV and YrA. No virulence was detected on plants with genes Yr4, Yr5, Yr10, Yr15, Yr24 and YrSP.

Keywords: wheat, stripe rust, resistance cultivars, aggressive, virulence

Introduction

Yellow rust (stripe), caused by Puccinia striiformis, is one of the most important diseases that influence and reduce wheat production. Yellow rust affected 20 to 40

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percent of wheat production from 1999 to 2004 in Central Asia (Morgounov et al., 2004). Wheat (Triticum aestivum L.) is a strategic cereal with a direct role in the provision of food for humans and an indirect role in the production of animal protein. Wheat breeding programmes aim to improve yield potential, disease resistance and wheat quality. The most practical and environmentally friendly means of protecting wheat crops against rust diseases is the use of resistant cultivars (FAO, 2014). Stripe rust can cause 100% loss of yield if infection occurs very early and the disease continues to develop through the growing season (Chen, 2005). Afshari (2013) has reported that 41 races were determined from 104 isolates in greenhouse conditions from 2008 to 2010 in Iran. Races 6E6A+, 6E10A+ and 6E0A+ were more common. Race 0E0A+ was less aggressive than races 166E158A+ and 134E158A+ with virulence on 11 known genes. "Virulence on plant/s with gene/s Yr1, Yr2, Yr4, Yr6, Yr7, Yr8, Yr9, Yr10, Yr25, Yr27, YrSU, YrSD, YrND, Yr3, Yr2+, Yr6+, Yr9+, Yr7+, YrCV and YrA was detected". "The majority of isolates with high frequency (more than 70%) displayed virulence on plant/s with Yr2, Yr7, Yr9 and YrA genes. No virulence was detected on plant/s with Yr3, Yr5 and YrSP. In greenhouse tests, the frequency of virulence to wheat genotypes with Yr1, Yr4, Yr10, YrCV (32+) and YrSD gene was less than 7%. Frequency of virulence to other wheat genotypes was between 8 and 100%" (Afshari, 2013).

Pornamazeh collected 37 isolates of yellow rust pathogen from the most important wheat-growing areas in Iran. The results showed that the race 166E254A+,Yr27+ with 62.50% pathogenesis was the most aggressive race from Torogh (Mashhad), and the race 6E134A+ with 33.34% pathogenesis factor was the weakest race from Zarghan1. According to these results, for all the plants containing genes Yr2, Yr6, Yr7, Yr9, Yr18, YrA virulence was observed from all isolates. Plants containing genes Yr1, Yr4, Yr5, Yr10, Yr15, YrSU were effective against all isolates (Pornamazeh et al., 2013). Dalvand et al. (2013) conducted a survey over two years (2010–2011 and 2011–2012) in Khuzestan province in the south of Iran (warm zone) using a trap nursery, which included 41 lines of standard sets of stripe rust with different genes and Bolani and morocco as susceptible checks; data showed that virulence in genes included Yr6, Yr7, Yr9, Yr27 and YrA. For genes Yr1, Yr3, Yr4, Yr5, Yr8, Yr10, Yr15, YrCV, YrND, YrSU, YrSP and YrSD virulence was not detected. In Turkey in 2009-2011, experiments concluded that Puccinia striiformis is virulent in Yr2, Yr6, Yr7, Yr9, Yr18, Yr27, Yr28, and Yr31 and avirulent in Yr1, Yr3, Yr4, Yr5, Yr8, Yr10, Yr15, Yr17, YrSP, YrCV (Tekdal et al., 2012). Again, in Turkey, 54 isolates were differentiated into 27 and 45 races using 20 World and 20 US wheat differentials, respectively. None of the isolates were virulent on Yr5, Yr10, Yr15 or Moro, which carries Yr10 and YrMor (Sharma-Poudyal et al., 2013). Due to the possible emergence of new races of yellow rust and disease spreading quickly worldwide which could cause enormous damage, it is essential to manage breeding programmes

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Race Analysis of Puccinia Striiformis f.sp. Tritici in Iran 29

based on genetical knowledge of the pathogenicity of pathogens and relationships between pathogen and host. As has been proven, many resistance genes have prevented epidemics of yellow rust. Such a resistance gene can be identified by persistent monitoring and examining yellow rust. This study was carried out to identify genes in wheat resistant to stripe rust, in order to prevent epidemics of yellow rust and to use these genes in breeding programmes.

Materials and methods

In this research, various isolates of yellow rust from infected wheat leaves were collected from 26 regions of Iran (Fig. 1) and transferred to the Seed and Plant Improvement Institute, Karaj-Iran. Each sample was then inoculated in the seedlings with susceptible Bolani. In order to determine the pathogenicity of pathogens and gene analysis, 56 differential sets and isogenic lines of wheat were used (Table 1). Inoculation was with a mixture of yellow rust urediniospores and industrial oil Tween 20 and spread by air-pump into Bolani leaves surface. Inoculation was done in 12 step of the Zadoks scale (Zadoks et al., 1974). In order to germinate spore, pots were kept in a cold-room for 24 hours at 10 °C and 100 per cent humidity. The pots were then transferred to a greenhouse with 60 to 70 per cent humidity, at 18°C and 16000 lux for 16 hour days. After 17 days, types of infection were noted by the MacNeal method on a scale of 0 to 9 (McNeal et al., 1971). Determination of isolate race was carried out according to the study described by Johnson et al. (1972) (Table 2).

Table 1. Differential sets and the isogenic lines of wheat No. Differential set

World Series Resistance

genes No. Differential set

World Series Resistance genes

1 Chinese 166 Yr1 28 Yr6/6*Avocet ’S’ Yr6 2 Lee Yr7 29 Yr7/6*Avocet ’S’ Yr7 3 Heines kolben Yr2,Yr7 30 Yr8/6*Avocet ’S’ Yr8 4 Vilmorin 23 Yr3 31 Yr9/6*Avocet ’S’ Yr9 5 Moro Yr10 32 Yr10/6*Avocet ’S’ Yr10 6 Strubes Dikkopf YrSd 33 Yr15/6*Avocet ’S’ Yr15 7 Suwon 92/omar YrSU 34 Yr17/6*Avocet ’S’ Yr17 8 Clement Yr2,Yr9,+ 35 Yr18/6*Avocet ’S’ Yr18 9 Triticum spelta

var.album Yr5 36 Yr24/6*Avocet ’S’ Yr24

European Series

37 Yr26/6*Avocet ’S’ Yr26

10 Hybrid 46 Yr4 38 Yr27/6*Avocet ’S’ Yr27

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11 Reichersberg 42 Yr7+ 39 Yr32/6*Avocet ’S’ Yr32 12 Heines Peko Yr2,Yr6,+ 40 YrSP/6*Avocet ’S’ YrSP 13 Nord desprez YrND 41 Jupateco 73R 14 Compare . Yr8 42 Jupateco 73S 15 Carstens V YrCV 43 Avocet’R’’ YrA 16 Spalding Profic YrSP 44 Avocet’S’ 17 Heines VII Yr2+ 45 Bolani Australian

Series 46 Fielder Yr6,Yr20

18 Avocet ‘R’ YrA 47 Thatcher Yr7 19 Kalyansona Yr2 48 Lemhi Yr21 20 Trident Yr17+sr38 49 Tp1295 Yr25 21 Yr 15/6* Avocet

S Yr15 50 Yr27/6*Avocet’s’ Yr27

22 Hugenoot Yr25 51 Ciano79 Yr27 23 Selkirk Yr27 52 Opata85 Yr27+Yr1

8 24 Federation *4/

kavakaz Yr9 53 Avocet Yr28 Yr28

25 Federation

54 Lalbahador/pavon Yr29 26 Yr 1/6* Avocet

’S’ Yr1 55 Avocet-

YrA*3/pastor Yr31

27 Yr5/6*Avocet ’S’

Yr5 56 Pastor Yr31+APR

Results and discussion

In this research 26 isolates of yellow rust that were collected from different parts of Iran (Fig. 1) were inoculated separately in 56 differential sets and isogenic lines of wheat in greenhouse conditions. Virulence was detected against plants with gene/s Yr1, Yr2, Yr3, Yr6, Yr7, Yr8, Yr9, Yr18, Yr21, Yr25, Yr26, Yr28, Yr29, Yr31, Yr32, YrSU, YrND, YrCV and YrA, which were more common in Iran. The research found no virulence against plants with genes Yr4, Yr5, Yr10, Yr15, Yr24 and YrSP and they were resistant to all 26 isolates of yellow rust (Table 2). Pathotype analyses by Prashar revealed that yellow rust in the wheat population of India is avirulent against Yr5, Yr10, Yr11, Yr12, Yr13, Yr14, Yr15, Yr24, Yr26, YrSp and YrSk (Prashar et al., 2015). Consideration of the reactions of differentials and isogenic lines to the P. striiformisf.sp. tritici populations during the six years from 2006 to 2012 determined virulence against Yr2, Yr6, Yr7, Yr8, Yr9, YrSU, Yr17, Yr22, Yr23, Yr24, Yr25, Yr26, YrA and Yr27 and avirulence against Yr1,Yr2+, Yr3V, Yr3a, Yr4a, Yr4, Yr5, Yr7+, Yr10, Yr15,Yr16, YrCV, YrSD and YrND (Safar Ali Safavi et al., 2013). Studies of

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Race Analysis of Puccinia Striiformis f.sp. Tritici in Iran 31

pathogenicity indicated that effective genes included Yr1, Yr2+, Yr3, Yr4, Yr5, Yr10, Yr15, Yr24, Yr26, YrSP, YrND, YrSD and YrSU (Omrani et al., 2013). This research indicates that the Yr1 gene has lost its resistance over the years and could not be used as a resistance gene in most regions in Iran. Although Yr3 was identified as a resistance gene in previous research, it has virulence in 6 regions of Iran. Compatible infection types 7 to 9 against Yr1, Yr2, Yr3, Yr4, Yr6, Yr7, Yr9, Yr17, Yr25, Yr32, and YrSp were recorded in samples collected in 2014 from Thrace and Sakarya in Turkey (Mert et al., 2014).

In our research, plants with gene Yr24 were resistant to all 26 isolates. Other work by Dalvand (et al., 2013) showed that plants with the genes YrA, Yr6, Yr7, Yr9, and Yr27 were susceptible, indicating that these genes are not effective. Results in Pakistan revealed that stripe rust resistance genes Yr3, Yr5, Yr10, Yr15, Yr26, YrSP and YrCV were resistant, while Yr18 displayed moderate susceptibility at all locations. Genes YrA-, Yr2, Yr6, Yr7, Yr8, Yr9, Yr17, Yr27 and gene combinations Opata (Yr27+Yr18) and Super Kauz (Yr9, Yr27, Yr18) were found to be susceptible (Bux et al., 2011).

Races collected from Neishabour (Eshgh Abad) and Kermanshah were less aggressive in this research and races from Eslam Abad, Fars and Mashhad were the most aggressive. Soweizy (et al., 2016) reported no pathogenicity recorded in plants carrying the genes Yr5, Yr10, Y15, and YrSP. Race 132E156A+, Yr27 of Ahvaz 3 and race 2E2A+ of Mashhad were identified as the most and least aggressive races, respectively. However, the first detection of Yr24 virulence in P. striiformis populations on Chuanmai 42 was by Liu et al. (2010).

Race 6E6A+,Yr27 and 7E22A+,Yr27 which were the least aggressive races in this research have virulence against plants with gene/s Yr1, Yr2 ,Yr6, Yr7,Yr8,Yr9, Yr17, Yr18,Yr27,YrA, and races 206E182A+,Yr27, 207E190A+, Yr27 and 231E150A+,Yr27 which were the most aggressive races have virulence against plants with genes Yr1, Yr2 ,Yr6 , Yr7 Yr8, Yr9, Yr18, Yr25, Yr26, Yr27, Yr32, YrA, YrSD, YrSU. The results of monitoring the virulence factors of wheat yellow rust are shown in Table 2.

An investigation of the pathotypes 6E134A+, 6E142A, 6E6A+, 134E142A, 6E158A+, 134E130A+, 6E22A+, 6E130A+ recorded greater frequency of virulence against plants containing the genes YrA, Yr32, Yr9+, Yr7+, Yr2+, YrSP, YrSD, Yr24, Yr9, Yr8, Yr7, Yr6, Yr2 and the least frequency against the genes YrSU, YrCV, Yr1, Yr10, Yr5, Yr4, Yr3 in Iran (Afshari, 2008).

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32 Serpoush et al.

Fig. 1. Map of Iran, showing the locations of yellow rust regions.

Table 2. Avirulence/virulence of wheat yellow rust isolates

Avirulence/ Virulence genes Race Region No.

Yr1, Yr3, Yr10, YrSD, YrSU, Yr5, Yr4, YrND, YrCV,YrSP, Yr15, Yr24, Yr26 , Yr32, Yr28, Yr31,Yr31+APR/ Yr7, Yr2,

Yr9, Yr2.Yr6.+ , Yr8, YrA, Yr17+sr38, Yr25,Yr27, Yr6, Yr18, Yr6.Yr20, Yr21, Yr29

134E150A+, Yr27

Ardebil 1

Yr1, Yr3, Yr10, YrSd, Yr5, Yr4, YrSP, Yr15, Yr5, Yr24, Yr26 ,YrSP/ Yr7, Yr2.Yr7, YrSU, Yr2Yr9+, Yr7+, YrND, Yr8 , Yr2+, YrA, Yr17+sr38, Yr25, Yr27, Yr9, Yr6, Yr7, Yr8, Yr17, Yr18, Yr32, Yr6Yr20, Yr21, Yr28, Yr29, Yr31,

Yr31+apr

198E154A+, Yr27

Zarghan 2

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Race Analysis of Puccinia Striiformis f.sp. Tritici in Iran 33

Yr1, Yr3, Yr10, YrSd, YrSU, Yr5, Yr4, YrND, Yr8, YrCV, YrSP, Yr15, Yr27, Yr8, Yr17, Yr18, Yr24, Yr32, Yr28,

Yr29, Yr31+apr/Yr2Yr7, Yr2Yr9+, Yr2Yr6+, Yr2+, YrA, Yr25, Yr9, Yr6, Yr26, Yr7, Yr21, Yr31, Yr2

134E134A+, Yr27

Sari 3

Yr1, Yr3, Yr10, YrSd, YrSU, Yr2Yr9+, Yr5, Yr4, YrND, Yr8, YrCV, YrSP, Yr15, Yr27, Yr24, Yr26,Yr8 ,Yr32/ Yr7,

Yr2Yr7, Yr7+, Yr2Yr6+, Yr2+, YrA, Yr2, Yr17+sr38, Yr25, Yr9, Yr6, Yr17, Yr18, Yr6,Yr20, Yr21, Yr25, Yr27+Yr18,

Yr28, Yr31, Yr31+apr ,Yr29

6E134A+ Mashhad 4

Yr1,Yr10,Yr3,YrSD,Yr2Yr9+,Yr5,Yr4,YrND,YrCV,YrSP,Yr15,Yr24/Yr7,Yr2,YrSU,Yr2Yr6+,Yr8,YrA,Yr17+Sr38,Yr25,

Yr27,Yr9,Yr6,Yr17,Yr18,Yr26,Yr32

70E150A+,Yr27

Kermanshah

5

Yr3,Yr10,YrSD,YrSU,Yr2Yr9+,Yr5,Yr4,YrCV,YrSP,Yr15,Yr24/Yr1,Yr7,Yr2,Yr2Yr6+,YrND,Yr8,YrA,Yr17+Sr38,Yr25,

Yr27,Yr9Yr6,Yr17,Yr18,Yr26,Yr32

7E158A+,Yr27

Karaj 6

Yr1,Yr10,YrSD,Yr5,Yr4,YrND,YrSp,Yr15,Yr24/ Yr7,Yr2,Yr3,YrSU,Yr2Yr9+,Yr2Yr6+,Yr8,YrCV,YrA,Yr17+

Sr38,Yr25,Yr27,Yr9,Yr6,Yr7,Yr17,Yr18,Yr26,Yr32

206E182A+,Yr27

Eslam abad

7

Yr1,Yr10,Yr5,Yr4,YrND,YrCV,YrSp,Yr15,Yr24/Yr7,Yr2,Yr3,YrSD,Yr2Yr9+,Yr2Yr6+,Yr8,YrA,Yr17+Sr38,Yr25,Yr27,Y

r9,Yr6,Yr7,Yr17,Yr18,Yr26,Yr32

110E150A+,Yr27

Zarghan2 8

Yr1,Yr10, YrSD,Yr2Yr9+,Yr5,Yr4,YrSP,Yr15,Yr24 / Yr7,Yr2,Yr3,YrSU,Yr2Yr6+,YrND,Yr8,YrCV,YrA,Yr17+Sr

38,Yr25,Yr27,Yr9,Yr6,Yr17,Yr18,Yr26,Yr32

78E190A+,Yr27

Moghan 9

Yr1,YrSD,YrSU,Yr2YR9+,Yr5,Yr4,YrND,YrCV,YrSP,Yr15/Yr7,Yr2,Yr3,Yr10,Yr7+,Yr2Yr6+,Yr8,YrA,Yr17Sr38,Yr25,

Yr27,Yr9,YrYr10,Yr17,Yr18,Yr24,Yr26,Yr32,Yr6Yr20

30E150A+,Yr27

Mashhad2

10

Yr1,Yr10,Yr2Yr9+,Yr5,Yr4,YrNd,YrCV,YrSP,Yr15,Yr24/Yr7,Yr2,Yr3,YrSD,YrSU,Yr7+,Yr2Yr6+,Yr8,Yr2+,YrA,Yr17+

Sr38,Yr25,Yr27,Yr9,Yr6,Yr8,Yr17,Yr18,Yr26,Yr32

110E150A+,Yr27

Brojerd 11

Yr1,Yr3,Yr10,Yr2Yr9+,Yr5,Yr4,Yr2Yr6+,YrND,Yr8,YrCV,YrSP,Yr17+Sr38,Yr15,Yr25,Yr27,Yr17,Yr24,Yr26/Yr7,Yr2,YrSD,YrSU,Yr7+,Yr2+,YrA,Yr9,Yr6,Yr18,Yr32,Yr6Yr20

102E130A+

Moghan2 12

Yr1,Yr7,Yr3,Yr10,YrSU,Yr2Yr9+,Yr5,Yr4,Yr7+,Yr2Yr6+,YrND,Yr8,YrCV,YrSP,YrA,Yr17+Sr38,Yr15,Yr25,Yr9,Yr6,Yr

17,Yr18,Yr24,Yr26,Yr32/Yr2,YrSD,Yr2+,Yr27,Yr6Yr20

36E128A-,Yr27

Neishaboor (saad abad)

13

Yr1,Yr3,Yr10,YrSd,YrSU,Yr2Yr9+,Yr5,Yr4,YrCV,YrSP,Yr15,Yr27,Yr9,Yr24,Yr26,Yr32/Yr7,Yr2,Yr7+,Yr2Yr6+,YrND,Yr8,Yr2+,YrA,Yr17+Sr38,Yr25,Yr6,Yr8,Yr9,Yr17,Yr18,Yr6

Yr20

6E158A+ Zarghan3 14

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34 Serpoush et al.

Yr3,Yr10,YrSD,Yr2Yr9+,Yr5,Yr4,YrCV,YrSP,Yr2+,Yr17+Sr38Yr15,Yr24,Yr26/Yr1,Yr7,Yr2,YrSU,Yr7+,Yr2Yr6+,YrN

d,Yr8,YrA,Yr25,Yr27,Yr9,Yr6,Yr8,Yr18,Yr32,Yr6Yr20

71E30A+,Yr27

Khoram abad

15

Yr3,Yr10,YrSD,YrSU,Yr2Yr9+,Yr5,Yr4,YrND,YrCV,YrSP,Yr2+,Yr17+Sr38,Yr15,Yr25,Yr24,Yr26,Yr32/Yr1,Yr7,Yr2,Yr7+,Yr2Yr6+,Yr8,YrA,Yr27,Yr9,Yr6,Yr8,Yr9,Yr17,Yr18,Yr

6Yr20

7E22A+,Yr27

Kermanshah2

16

Yr10,YrSd,Yr5,Yr4,YrSP,Yr15,Yr24/Yr1,Yr7,Yr2,Yr3,YrSU,Yr2Yr9+,Yr7+,Yr2Yr6+,YrND,Yr8,YrCV,Yr2+,YrA,Yr17+Sr38,Yr25,Yr27,Yr9,Yr6,Yr8,Yr17,Yr18,Yr26,Yr32,Yr6Yr20

207E190A+,Yr27

Fars 17

Yr3,Yr10,Yr5,Yr4,YrNd,YrCV,YrSP,Yr17+Sr38,Yr15,Yr17,Yr24/Yr1,Yr7,Yr2,YrSD,YrSU,Yr2Yr9+,Yr7+,Yr2Yr6+,Yr8

,Yr2+,YrA,Yr25,Yr27,Yr9,Yr6,Yr18,Yr26,Yr32,Yr6Yr20

231E150A+,Yr27

Mashhad3

18

Yr1,Yr3,Yr10,YrSD,Yr5,Yr4,YrND,YrCV,YrSP,Yr15,Yr24,Yr32/Yr7,Yr2,YrSU,Yr2Yr9+,Yr7+,Yr2Yr6+,Yr8,Yr2+,YrA,

Yr17+Sr38,Yr25,Yr27,Yr9,Yr6,Yr8,Yr26,Yr6Yr20

198E150A+,Yr27

Sirjan 19

Yr1,Yr3,Yr10,YrSD,YrSU,Yr5,Yr4,YrND,YrCV,YrSP,Yr15,Yr24,Yr32/Yr7,Yr2,Yr2Yr9+,Yr7+,Yr2Yr6+,Yr8,Yr2+,YrA,

Yr17+Sr38,Yr25,Yr27,Yr9,Yr6,Yr17,Yr18,Yr26,Yr6Yr20

134E150A+,Yr27

Zarghan4 20

Yr1,Yr3,Yr10,YrSD,YrSU,Yr2Yr9+,Yr5,Yr4,YrNd,YrCV,YrSP,Yr15,Yr24,Yr26,Yr32/Yr7,Yr2,Yr7+Yr2Yr6+,Yr8,Yr2+,

YrA,Yr17+Sr38,Yr25,Yr27,Yr9,Yr6,Yr17,Yr18,Yr6Yr20

6E150A+,Yr27

Neishabour (mian

jolge) 21

Yr3,Yr10,Yr2Yr9+,Yr5,Yr4,YrND,YrCV,YrSP,Yr15,Yr24,Yr26,Yr27,Yr32/Yr1,Yr7,Yr2,YrSD,YrSU,Yr7+,Yr2Yr6+,Yr8,

Yr2+,YrA,Yr17+Sr38,Yr25,Yr9,Yr6,Yr17,Yr18,Yr6Yr20

103E150A+

Sarvdasht

22

Yr1,Yr3,Yr10,YrSD,YrSU,Yr2Yr9+,Yr5,Yr4,YrNd,YrCV,YrSP,Yr15,Yr24,Yr32/Yr7,Yr2,Yr7+,Yr2Yr6+,Yr8,Yr2+,YrA,

Yr17+Sr38,Yr25,Yr27,Yr9,Yr6,Yr17,Yr18,Yr6Yr20

6E150A+,Yr27

Torbat heidarie

23

Yr1,Yr3,Yr10,YrSd,YrSU,Yr2Yr9+,Yr5,Yr4,YrND,Yr8,YrCV,YrSP,Yr2+,Yr17+Sr38,Yr15,Yr8,Yr17,Yr24,Yr26,Yr32/Yr7,Yr2,Yr7+,Yr2Yr6+,YrA,Yr25,Yr27,Yr9,Yr6,Yr18,Yr6Yr2

0

6E6A+,Yr27

Neishabor (Eshgh

abad) 24

Yr1,Yr3,Yr10,YrSD,YrSU,Yr5,Yr4,YrND,YrCV,YrSP,Yr15,Yr24,Yr26,Yr32/Yr7,Yr2,Yr2Yr9+,Yr7+,Yr2Yr6+,Yr8,Yr2+,YrA,Yr17+Sr38,Yr25,Yr27,Yr9,Yr6,Yr17,Yr18,Yr6Yr20

6E150A+,Yr27

Pol-dokhtar

25

Yr1,Yr10,YrSD,YrSU,Yr2Yr9+,Yr5,Yr4,YrND,YrCV,YrSP,Yr17+Sr38,Yr15,Yr17,Yr24/Yr7,Yr2,Yr3,Yr7+,Yr2Yr6+,Yr8,Yr2+,YrA,Yr25,Yr27,Yr9,Yr6,Yr18,Yr26,Yr32,Yr6Yr20

14E150A+,Yr27

Brojerd 26

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Race Analysis of Puccinia Striiformis f.sp. Tritici in Iran 35

Conclusion

In view of the emergence of new races of yellow rust it is important to control and monitor resistance genes. In this research, we observed that plants with the genes Yr4, Yr5, Yr10, Yr15, Yr24 and YrSP are totally resistant in all regions of Iran, and it is possible to use them in wheat breeding programmes in Iran.

Acknowledgements

Thanks to the Seed and Plant Improvement Institute for providing facilities for this research.

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resistance to stripe rust and the virulence of Puccinia striiformis f. sp. tritici in Pakistan. Vol. 10(28), pp. 5489-5495, 20

Chen, X. M. (2005). Epidemiology and control of stripe rust [Puccinia striiformis f. sp. tritici] on wheat. Can. J. Plant Pathol. 27:314-337.

Dalvand, M., Afshari, F., & Aeini, M. (2013). Virulence survey of Puccinia striiformis, the causal agent of wheat yellow rust, by trap nursery 2010–2012. The causal agent of wheat yellow rust, by trap nursery 2010 to 2012, Archives of Phytopathology and Plant Protection. 47:12, 1508-1513

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Liu, T. G., Peng, Y., Chen, W. Q., & Zhang, Z. Y. (2010). First Detection of Virulence in Puccinia striiformis f. sp. tritici in China against Resistance Genes Yr24 (=Yr26) Present in Wheat Cultivar Chuanmai 42. Plant disease, 94.9: 1163-1163

Mert, Z. K., Nazari, E., Karagöz, K., Akan, I., Öztürk, A., & Tülek, A. (2014). The First Incursion of the Warrior Race of Wheat Stripe Rust (Puccinia striiformis f. sp. tritici) to Turkey in 2014. Plant disease; 100, 2; 528-528

Morgounov, A., Yessimbekova, M., Rsaliev, S., Baboev, S., Mumindjanov, H., & Djunusova, M. (2004). High yielding winter wheat varieties resistant to

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yellow and leaf rust in Cereal Asia. In Proceedings of the 11th International Cereal Rust and Powdery Mildews Conference 22-27August 2004.

Omrani, A., Khodarahmi, M., & Afshari, F. (2014). Reaction of some wheat cultivars and breeding lines to Puccinia striiformis f. sp. tritici hot races in Iran. Archives of Phytopathology and Plant Protection. 47:9, 1136-1145

Pornamazeh, P., Afshari, F., & Khodarahmi, A. (2013). The genetics of pathogenicity of Puccinia striiformis f. sp. tritici the causal agent of wheat yellow rust disease in Iran, Archives of Phytopathology and Plant Protection, 46:12, 1497-1507

Prashar, M., Bhardwaj, S. C., Jain, S. K., & Gangwar, O. P. (2015). Virulence diversity in Puccinia striiformis f.sp. tritici causing yellow rust on wheat (Triticum aestivum) in India. Indian Phytopath. 68:2, 129-133

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Sharma-Poudyal, D., Chen, X. M., Wan, A. M., Zhan, G. M., Kang, Z. S., Cao, S. Q., Jin, S. L., Morgounov, A., Akin, B., Mert, Z., Shah, S. J. A., Bux, H., Ashraf, M., Sharma, R. C., Madariaga, R., Puri, K. D., Wellings, C., Xi, K. Q., Wanyera, R., Manninger, K., Ganzález, M. I., Koyda, M., Sanin, S., & Patzek, L. J. (2013). Virulence characterization of international collections of the wheat stripe rust pathogen, Puccinia striiformis f. sp.tritici. Plant Disease, 97: 379-386.

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Khazar Journal of Science and Technology Volume 3 №1 2019, 37-38 © Khazar University Press 2019 DOI: 10.5782/2520-6133.2019.3.1.37

37

Do CRISPR or CRISPR-like Systems and Sequences Exist in Human Mitochondria or Evolutionary Disappeared in Human Cells?

Allahverdi Shahveranov

Genetic Resources Institute, Azerbaijan National Academy of Sciences Corresponding author: [email protected]

CRISPR (clustered regularly interspaced short palindromic repeats) and related genes (cas) or possible CRISPR-like gene transfer systems and related genes (especially short palindromic repeats are considered) may exist in human mitochondria (especially in hepatocytes and T-lymphocytes). Hereby, each type of CRISPR-Cas systems including I, II, III and IV or similar systems are considered. We should regard that human mitochondria is considered as the predecessor of the bacterium that is absorbed into mammalian cells by the anaerobic eukaryotic organism that is embedded in our cells as a result of evolution. (Cox et al., 2018; Dyall et al., 2014; Tielens et al., 2012; Lan et al., 2006) Human mitochondrial DNA (mtDNA) integrity might be also protected by CRISPR-Cas systems or CRISPR-like systems. Systems such as base excision repair (BER), mismatch repair (MMR), single-strand break repair (SSBR), microhomology-mediated end joining (MMEJ), and possibly homology recombination dependent repair (HRR) are still insufficient for understanding mtDNA integrity completely, and not inefficinet for designing novel detailed specific mapping of the protection mechanisms of mtDNA. Therefore, I want to mention that repeated CRISPR or CRISPR-like sequences should be considered and investigated in further mtDNA investigations. Furthermore, we also need to explore CRISPR or CRISPR-like systems in the human cell nucleus. Because the genes encoding mitochondrial proteins which responsible for the repair of mtDNA damage are nuclear-origin.

One of the mechanisms of protection systems against hepatitis virus types in hepatocytes may belong to CRISPR or CRISPR-like systems. Currently, certain antiviral protection systems for hepatocytes are not fully understood and not deeply investigated, and consequential investigations are needed for obtaining the precise results. CRISPR or CRISPR-like systems will play a major role in the development of more efficient diagnostic and therapeutic protocols and molecular biologic mapping of diseases related to genetical and epigenetical damages for next decades. Certainly, it is also important to consider ethical rules when exploring and applying

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38 Shahveranov

genome editing systems such as CRISPR or CRISPR-like system in human cells. As a result, it should be noted that CRISPR or CRISPR-like systems in human cells need to investigate, urgently. If the positive results indicate based on my idea, I will be so grateful for being part a role in encouraging further novel worldwide genomic and epigenomic studies and designing novel scientific approaches.

References: Coxa, C. J., Fostera, P. G., Hirtb, R. P., Harrisb, S. R., & Embley T. M. (2008). The

archaebacterial origin of eukaryotes. PNAS 105, 20356–20361 doi:10.1073/pnas. 0810647105.

Dyall, S. D., Brown, M. T., & Johnson, P. J. (2004). Ancient invasions: From endosymbionts to organelles. Science 304, 253–257, doi:10.1126/science.1094884.

Tielens, A. G., Rotte, C., van Hellemond, J. J., & Martin, W. (2012). Mitochondria as we don't know them. Trends in Biochemical Sciences 27, 564–572, doi:10.1016/S0968-0004(02)02193–X.

Lane, N. (2006). Power, Sex, Suicide: Mitochondria and the Meaning of Life. Oxford: Oxford University Press.

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Khazar Journal of Science and Technology Volume 3 №1 2019, 39-45 © Khazar University Press 2019 DOI: 10.5782/2520-6133.2019.3.1.39

39

Conceptual Approach of Regional Problem of the Global Climate Change

Rustam Rustamov

eiLink Research and Development Center of Khazar University

Abstract

The Earth’s climate system is the consequence of a complex interplay between external solar forces and the internal interactions of atmosphere, oceans, land surface, biosphere and cryosphere. Human activity is a potential factor influencing change in the global system by altering the chemical composition of atmospheric concentrations of powerful greenhouse gases, mainly CO2 and CH4. How and what should be undertaken to minimize such impact? Space-based technology is, to a point, able to accurately observe and sense the whole Earthly system and to understand the processes involved in the Earth’s climate. This suggests the following projects; to document and understand the interrelation between Sun and Earth as an external force acting on the Earth’s climate and also to seek a better understanding of the Earth’s intricately linked internal processes including those of global water, energy and carbon cycles. Together, advances in computing and information systems technologies, and modern techniques in data assimilation, diagnostic and prediction models, all provide a powerful combination of tools for understanding the Earth’s system and applying knowledge and tools to the management of natural resources and mitigation of natural hazards. Cooperation can likely be developed within the framework of existing programmers. There is no doubt that contributions to climate change are on a global scale, necessitating the engagement of states and international institutions: the Global Monitoring for Environment and Security (GMES) programme - a joint initiative of the European Commission (EC) and the European Space Agency (ESA), is designed to establish a European capacity for the provision and use of operational information for global monitoring of the environment. And the United Nations Platform for Space-based Information for Disaster Management and Emergency Response – UN-SPIDER - is another opportunity for the enhancement of the above-mentioned projects for the establishment of international cooperation.

Keywords: Space technology, climate change, global monitoring system, GIS, data processing

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40 Rustamov

Introduction

Future applications in space promise revolutionary improvements to the quality, style and culture of our lives. A fundamental element of the National Space Society’s vision is its commitment to promoting the use of space resources for the benefit of humanity. Undoubtedly, many resources will be discovered in the future on asteroids, planets and, possibly, the moons of our solar system and will be exploited. However, resources in space are not limited to mining materials and lunar soil. Space itself is a valuable resource, as a host for satellites able to see and efficiently communicate with large areas of the Earth’s surface (Gérardine Meishan, 2008).

This resource has spawned many applications of space systems that, every day, improve life on Earth. Some of the space programme’s greatest success stories offer even greater promise for the future. There is an important factor to keep in mind when we talk about the use of space to improve life on Earth (Rustamov et al., 2011).

Space’s contributions to our day-to-day needs are sometimes referred to as “space spin-offs”. This term more appropriately describes ancillary uses of technologies developed for space systems, rather than direct use of the systems themselves for their original purpose. Direct uses of space technology are more accurately called “space applications”.

The fact is that selection of facilities for high accuracy and efficient information towards an understanding of the processes is very important to the achievement of the desired results. This is especially so when a wide range of data sources need to be assembled quickly and processed for due clarification of consequences occurring.

Here, the advances in space technology become useful (Ashumova et al. 2018; Okon, 2017) for studies of climate change within a regional framework.

Results and discussion

Approach Area

It following options should be taken into consideration:

Space applications are a perfect tool for the development of a methodology to monitor the processes of climate change;

Excellence of outcome, with high accuracy and relatively immediate feedback;

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Conceptual Approach of Regional Problem of the Global Climate Change 41

The integration of different countries’ capacities would be much improved on matters relating to: natural resources, investigations, natural disasters, climate change and security and many other vitally important problems.

It is important to establish and communicate how to implement the projects proposed for the achievement of best results. It may be suggested that there is clearly a great need for the collection of appropriate processed data and the main issue is to share the data with the communities who need it.

A further step could be to invite input from different countries, taking on the necessity of integration within the Black and Caspian Seas region as a testing ground for the identification of global climate changes. There is no doubt that the involvement of Central Asia countries would be highly desirable for a comprehensive understanding of climate change problems and global impacts, thus including a broad area of Earth and opening up a unique opportunity for data collection and processing, with excellent outcomes for the aims and targets proposed.

To adopt the approach suggested, engagement with the countries of the Black Sea and Caspian Sea region (Fig. 1) would be desirable.

Fig. 1. Black and Caspian Seas Region (BCSR) Partnership Countries

From these objectives:

An important issue of the global climate change investigation is to:

Identify causes of impacts on climate change and their consequences.

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42 Rustamov

Therefore: the necessity to collect and process appropriate data from space and field investigations and integrate it into a coordinated system of geographical information that is easily accessible for future needs.

Building a huge range of sources, particularly within the regional framework indicated, with valuable consequences for climate change.

The region has a wide range of industrial sectors, water pools, land use/land cover capacity, environmental impacts and many other aspects to make the BCSR a region of great interest for such a project.

Consideration should be given to the principal impacts of climate change. The region’s oil and gas infrastructure is one of these aspects impacting on natural processes. Fig. 2 reflects this infrastructure within the region.

Fig. 2. Map of the Oil and Gas infrastructure in the Black and Caspian Seas Region

The collection of appropriate data on a regional scale may be divided into two stages of country engagement. The first stage of the project’s implementation may be undertaken within the following countries: Turkey, Romania, Bulgaria, Ukraine, Georgia, Moldova, Azerbaijan, Kazakhstan, Uzbekistan, Kyrgyzstan, Tajikistan, Turkmenistan, Russia and Iran.

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Conceptual Approach of Regional Problem of the Global Climate Change 43

The second stage could embrace the following countries: Bosnia, Slovakia, Hungary, Greece, Croatia, Slovenia, Serbia, Belarus, Macedonia and others.

How to manage the process. Fig. 3 proposes a preliminary structure for project implementation.

Figure 3. Organizational Chart and Process proposed for the project

Project Implementation:

The following approach to project implementation may be considered:

Needs and opportunities in the development of global and regional reviews and research into global climate change: the creation of alliances between institutions to improve their effectiveness and coverage of urgent issues; the facilitation of

UN/BSO as a Project Manager

Black Sea Region Project Coordinator

Caspian Sea Region Project Coordinator

Azerbaijan Kazakhstan Turkmenistan NPoCs Russia Iran

Turkey Romania Bulgaria NPoCs Ukraine Georgia Moldova

Data collection

Data processing

Data analysis

Climate Change Dynamic Study features

Recommendations for decision-makers

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44 Rustamov

collaborative national research networks; stimulation to create new centres and networks where necessary; mobilization and coordination of resources.

Note that the eight Millennium Development Goals (MDG) were adopted by the international community as a framework for the development work of over 190 countries divided into ten regions. They are: eradicate extreme poverty and hunger, achieve universal primary education, promote gender equality and empower woman, reduce child mortality, improve maternal health, combat HIV/AIDS, malaria and other diseases, ensure environmental sustainability and develop a global partnership for development. The targets for implementation of each of these goals are reflected in the Millennium developments.

The Millennium goals and targets are sources for the successful investigations of problems relating to global climate change. We must strengthen global cooperation and redouble our efforts to reach the MDGs and advance the agenda for broader development. In the meantime, the use of existing international communities involved in global monitoring of the Earth and atmosphere studies, with applications of recent advances in space technologies and methods, may provide and develop extensive opportunities for study and exploration of the impacts of climate change.

Conclusion

It is evident that global phenomena demand the use and application of the tools available to contribute significantly to any solution. The problem of global climate change is one with a large scale of impact on human society and needs to be investigated with tools to collect and process the appropriate data. Thus, the use and application of space technology, particularly Remote Sensing methods and GIS technology needs to be applied to this purpose. Taking into account the seriousness of the problem it is my vision that success in these developments depends how well integrated and engaged are the countries in the various regions in data collection, processing and sharing between participants.

Reference Gérardine Meishan Goh, L. L. M. (2008). Legal Implications of Space Applications for

Global Climate Change, A Symposium jointly organized by the International Institute of Space Law and the European Centre for Space Law On the occasion of the 47th Session of the Legal Subcommittee of the United Nations Committee on the Peaceful Uses of Outer Space 31 March – 01 April, Report of the Symposium, p. 1-8.

Rustamov, B. R., Salahova, E. S., Hasanova, N. S., & Zeynalova, H. M. (2011). “Space Technology as a Tool in a Climate Change Monitoring System”, Climate Change.

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Conceptual Approach of Regional Problem of the Global Climate Change 45

Research and Technology for Adaptation and Mitigation, Edited by Juan Blanco and Dr. Houshan Kheradmand, Publishers INTECH, ISBN 979-953-307-277-6.

Ashumova, S. R., Musakhanova, L. I., Zeynalova, H. M., Mammadov, K. T., & Rustamov, R. B. (2018). Remote Sensing and the Geographical Information System as an Environment for Management of Engineering Activities, 14th International Conference on “Technical and Physical Problems of Electrical Engineering”, Nakhchivan State University, Nakhchivan, Azerbaijan, 15-17 October, ICTPE, Number 39, Code 09TAP01, pp. 193-197.

Okon, E. O. (2017). Climate Change: Space Technology and Climate-Resilient Development in Nigeria, International Journal of Social Sciences Perspectives ISSN 2577-7750 Vol. 1, No. 1, pp. 6-19 2017.

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Khazar Journal of Science and Technology Volume 3 №1 2019, 46-52 © Khazar University Press 2019 DOI: 10.5782/2520-6133.2019.3.1.46

46

The Dynamics of Some Carbon and Nitrogen Metabolism Enzymes in Daytime in Various Wheat

Genotypes during Drought Ulduza Gurbanova*, Shahniyar Bayramov, Irada Huseynova

Institute of Molecular Biology and Biotechnologies, 2A Matbuat ave., Baku, AZ1073, Azərbaycan

* corresponding author: [email protected]

Abstract PEPC plays a pivotal role in various metabolic processes in C3 plants including by providing intermediates for the Krebs cycle, maintaining intracellular pH and osmotic pressure, regulating the movement of stomatal guard cells, refixing CO2 formed by respiration, forming a carbon skeleton for lipid synthesis during grain development and nitrogen assimilation. Aspartate aminotransferase is essential to primary nitrogen assimilation, the transportation of reducing equivalents and the exchange of carbon and nitrogen resources among cellular subcompartments. Durum (Barakatli 95 and Garagylchyg 2) and bread wheat genotypes (Gobustan and Tale 38) cultivated in the experimental field of the Research Institute of Crop Husbandry were used as study materials. The high level of activity of PEPC and NAD-MDH during morning hours and a positive correlation between them in daytime suggest that, functioning mutually, these enzymes participate in the biosynthesis of malic acid.

Keywords: PEP-carboxylase, aspartate aminotransferase, NAD-malate dehydrogenase

Introduction

The annual production of wheat, which meets one fourth of global population demand for protein and calories, is ~700 million tons. This crop is cultivated on an area of 215 million ha, which is more than 16% of the world’s sowing fields. The main goals of contemporary and future biotechnology are to produce high-quality bread and biofuel, and to create varieties of wheat convenient for human consumption (Huppe and Turpin, 1994).

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Conceptual Approach of Regional Problem of the Global Climate Change 47

Playing a crucial role in carbon and nitrogen metabolism in plants, PEPC catalyses the irreversible carboxylation of phosphoenolepyruvate (PEP) and converts it into oxaloacetate (OAA) (Huppe and Turpin, 1994). The studies conducted are still not sufficient to elucidate the physiological role and regulatory mechanisms of C3 PEPCs (O’Leary et al., 2009). Currently, the role of PEPC in carbon and nitrogen metabolism in plants has not been studied sufficiently under in vivo conditions.

Aspartate aminotransferase (АsАТ, EC 2.6.1.1) plays an important role in primary nitrogen assimilation, the transportation of reducing equivalents and the exchange of carbon and nitrogen resources between cellular subcompartments (Gaufichon et al., 2016). Several isoforms of AsAT are localized in subcellular organoids – cytosol, chloroplasts, mitochondria and peroxisomes - of plants (Duff et al., 2011).

NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) participates in some metabolic processes, including the tricarboxylic acid and glyoxylate cycles, the synthesis of amino acids, glyconeogenesis and the exchange of metabolites between cytosol and subcellular organoids (Nunes-Nesi et al., 2005; Scheibe, 2004; Schertl and Braun, 2014).

Materials and methods

The Durum (Barakatli 95 and Garagylchyg 2) and bread wheat genotypes (Gobustan and Tale 38) used as study materials were cultivated in the experimental field of the Research Institute of Crop Husbandry located on the Absheron peninsula. Samples were taken at three-hourly intervals. The plant material was ground in the presence of quartz sand in a cooled mortar and pestle. The 100 mM Tris-HCl buffer used in homogenization contained 10 мМ MgCl2, 1мМ ethylenediaminetetraacetic acid (EDTA), 5 mM dithiothreitol (DTT), 2mM phenylmethanesulfonyl fluoride (PMSF) and 2% (w/v) insoluble polyvinylpyrrolidone (PVP).

PEPC activity was determined in a 1 ml reaction medium containing 50 mМ Tris-HCl (pH 8.0), 10 mM MgCl2, 2mM DTT, 10 mM NaHCO3, 0.2 mM NADH, 10 U/ml MDH, 10 mM PEP and 40 μl enzymatic extract. The reaction was initiated by adding a substrate (10 mM PEP) to the reaction medium (Pyankov et al., 2000).

AsAT activity was measured in a reaction medium containing 100 mM HEPES-KOH (pH 7.4) and 100 mM Tris-HCl (pH 8.5), 2 mM EDTA, 2.5 mM 2-oxoglutorate, 10μg/ml pyrodoxalphosphate, 10 mM DTT, 12 U/ml MDH, 0.2 mM NADH, 20μl leaf extract and 2.5 mM L-aspartate. The reaction was initiated by adding L-aspartate (Alfonso Brüggemann, 2012).

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48 Gurbanova

NAD-malate dehydrogenase activity was determined by adding a substrate (1 mM oxaloacetate) to a reaction medium containing 100 mM Tris-HCl (pH 9.0), 30 mM malate and 0.2 mM NAD.

Enzyme activity was determined by spectrophotometry (Ultrospec 3300 Pro, Amersham, USA). The measurements were taken in a 1ml cuvette, at 340 nm wavelength (Scheibe and Stitt, 1988).

Total protein assay: Total soluble protein was determined by spectrophotometry, using 0.12% Coomassie Brilliant Blue G-250 (Sedmak and Grossberg, 1977).

Results and discussion

Phosphoenolpyruvate carboxylase, aspartate aminotransferase and NAD-malate dehydrogenase activity was studied comparatively in durum and bread wheat genotypes with contrasting drought tolerance. In both variants of the durum wheat genotypes (Barakatli 95 and Garagylchyg 2) the highest level of PEPC activity was observed at 800. Whereas, in the bread wheat genotypes (Gobustan and Tale 38) the highest level of PEPC activity was recorded at 1700 in flag leaves. There were no significant changes in enzyme activity during morning and afternoon hours.

The highest level of AsAT activity was registered in the watered variant of the Barakatli 95 variety, the lowest level of activity in flag leaves of the stressed Garagylchyg 2 variety, which is drought sensitive (Fig. 1).

The highest level of AsAT activity in flag leaves of both variants of the drought-tolerant Gobustan variety was observed at 800. At 1100, enzyme activity declined by factors of 1.3 and 1.5 in watered and drought-exposed plant leaves, respectively. AsAT activity decreased gradually during the day, but began to increase from 1700, reaching the values observed in morning hours. Performing transamination in various cell compartments, AsAT catalyses the formation of aspartate and 2-oxoglutorate from glutamate and oxaloacetate (Fig. 2).

АsАТ plays an important role in primary nitrogen assimilation, the transportation of reducing equivalents and the exchange of carbon and nitrogen resources between cellular subcompartments.

The highest level of NAD-MDH activity was found in all samples taken from Barakatli 95 at 1700 with the exception of the drought-exposed variant. In the bread wheat varieties, the highest level of NAD-MDH activity was found in the morning hours (800 and 1100) of the day (Fig. 3). A gradual decrease in NAD-MDH activity

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Conceptual Approach of Regional Problem of the Global Climate Change 49

was observed in flag leaves of the drought-exposed Gobustan variety and watered Tale 38 variety during the day. Malate displayed a diurnal rhythm in the leaves of C3 plants.

Fig. 1. Dynamics of PEPC activity in flag leaves of durum and bread wheat genotypes during light phases of the day

0

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50 Gurbanova

Fig. 2. Dynamics of AsAT activity in flag leaves of durum and bread wheat genotypes during light phases of the day

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Conceptual Approach of Regional Problem of the Global Climate Change 51

Fig. 3. Dynamics of NAD-MDH activity in flag leaves of durum and bread wheat genotypes during light phases of the day

Conclusion

Increasing during periods of light, enzyme levels were highest at the end of the day and decreased during the night. Malate synthesis during periods of light is the result of the sequential functioning of PEPC and MDH. Thus, acting as a primary carboxylating enzyme, PEPC combines CO2 with PEP and forms 4-carbon, dibasic oxaloacetic acid.

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52 Gurbanova

Acknowledgments

This work was supported by the Science Development Foundation under the President of the Republic of Azerbaijan (Grant №: EİF-KETPL-2-2015-1(25)-56/35/3)

References Alfonso, S., & Brüggemann, W. (2012). Photosynthetic responses of a C3 and three C4

species of the genus Panicum with different metabolic subtypes to drought stress. Photosynthesis Research, v. 112, p. 175-91.

Duff, S. M., Qi, Q., Reich, T., Wu, X., Brown, T. (2011). A kinetic comparison of asparagine synthetase isozymes from higher plants. Plant Physiol Biochem v. 49, p. 251–256.

Gaufichon, L., Steven, J. R., & Akira, S. (2016). Asparagine metabolic pathways in Arabidopsis. Plant and Cell Physiology v. 57(4), p. 675–689.

Huppe, H., & Turpin, D. (1994). The integration of carbon and nitrogen metabolism in plant and algal cells. Annu. Rev. Plant Phys. Plant Mol. Biol., v.45, p.577–607.

Nunes-Nesi, A., Carrari, F., Lytovchenko, A., Anna, M. O., Marcelo E. L., George Ratcliffe R., Lee, J. S., & Alisdair, R. F. (2005). Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants. Plant Physiol., v. 137, p. 611–622.

O’Leary, B., Rao, S., Kim, J., & Plaxton, W. (2009). Bacterial-type Phosphoenolpyruvate Carboxylase (PEPC) Functions as a Catalytic and Regulatory Subunit of the Novel Class-2 PEPC Complex of Vascular Plants. J. Biol.Chem., v. 284, p. 24797-24805.

Pyankov, V. I., Voznesenskaya, E. V., Kuz'min, A. N., Ku, S. B., Ganko, E., Franceschi, R. V., Black, C. Jr., & Edwards, G. E. (2000). The occurrence of C3 and C4 photosynthesis in cotyledons and leaves of Salsola species (Chenopodiaceae). Photosynthesis Research, v. 63, p. 69-84.

Scheibe, R., & Stitt, M. (1988). A comparison of NADP-malate dehydrogenase activation, QA reduction and O2 reduction in spinach leaves. Plant Physiol Biochem., v. 26, p. 473–481.

Scheibe, R. (2004). Malate valves to balance cellular energy supply. Physiologia Plantarum v. 120, p. 21–26.

Schertl, P., & Braun, H. P. (2014). Respiratory electron transfer pathways in plant mitochondria. Front. Plant Sci. 5:163. doi: 10.3389/fpls.2014.00163.

Sedmak, J., & Grossberg, S. (1977). A rapid, sensitive and versatile assay for protein using Coomassie brilliant blue G-250 // Anal. Biochem, v. 79, p. 544-552 (1977).

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Khazar Journal of Science and Technology Volume 3 №1 2019, 53-57 © Khazar University Press 2019 DOI: 10.5782/2520-6133.2019.3.1.53

53

Sensitivity of Primary Tripsinized Cell Systems EYQ and FEC to the Fowl Pox Virus

Kubra Yusifova

Veterinary Scientific Research Institute, Azerbaijan Corresponding author: [email protected]

Abstract

Viral diseases like fowl pox create serious problems for industrial poultry. Fowl pox is a disease found in poultry worldwide and is caused by viruses of the family Poxviridae and the genus Avipoxvirus. The viruses causing fowl pox are distinct from each other but are antigenically similar. Possible hosts include chickens, turkeys, quail, canaries, pigeons and many other species of birds. It is known that the cultivation of a fowl pox virus in chicken embryo cell cultures is common practice for vaccine production. Many scientists use different local strains of fowl pox virus in attempts to improve the cultural vaccine against chicken fowl pox. The work described below demonstrates the possibility of cultivating and grouting a "Baku" strain of fowl pox virus in a primary cell culture of Japanese quail embryos.

It was discovered that the "Baku" bird strain of fowl pox virus has adapted to both cell systems. An increase in the titer of fowl pox virus was observed in both Japanese quail embryo cell culture and in that of chicken fibroblast embryos. It was found that the culture of Japanese quail embryo cells is a more effective tissue culture for vaccine production than chicken embryo cell culture because of its simplicity, economy, absence of extraneous contaminants and its stable biological properties. It was established that a cell culture of Japanese quail embryos is a promising basis for the creation of highly immunogenic specific prophylaxis against avian fowl pox.

Keywords: fowl pox, virus, primary cell culture, strains virus, vaccine

Introduction

Viral diseases like fowl pox create serious problems for industrial poultry. It is widespread and causes significant economic damage to poultry farms. It is already

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54 Yusifova

known that the cultivation of a fowl pox virus in chicken embryo cell cultures is common practice in preparing biomass for vaccine production. It is also known that the fowl pox virus has a cytopathic effect in cell culture, and with prolonged passage it becomes less contagious to the bird, but retains immunogenic properties. Last year’s anti-bird fowl pox cultural vaccines are not inferior in their immunogenicity to embryonic preparations (Patrushev, 1967; Sukhorukov, 2012; Zenov, 2010). Designed in Azerbaijan’s VCRI by F. B. Shirinov and A. N. Gojayev in 1985, the embryonic anti-chicken fowl pox vaccine from the "Baku" strain was grown on chicken embryos, and was thus imperfect in its production (Cherkezova, 2003; Elizbarashvili, 1987). The disadvantage of embryonic anti-bird fowl pox viral vaccines is the low level of infectious activity in chickens (Syurin, 1956; Tarasenko, 2003). In these circumstances, the search for fowl pox virus strains in birds adapted in a cell culture with highly infectious and immunogenic properties is appropriate for the production of modern drugs. Determining the sensitivity of primary cell cultures to fowl pox virus, monitoring the dynamics of the accumulation of poxvirus in a cell culture is appropriate in modern veterinary medicine.

In some countries, vaccines with attenuated virus strains are used in disease prevention: “Intervet” in the Netherlands, “Webster” in Australia, and others (Patrushev, 1967). Attenuated anti-bird fowl pox vaccines have been registered in Azerbaijan since 2002 to the present: in 2002 “Nobilis AE+Pox”, by the Dutch company Intervet International BV; in 2009 - “AviPro AE-Pox” and “AviPro Pox”, by the German company Lohman Animal Health International; in 2011 - “Gallivac AE+FP”, by the French company Merial; in 2014 “Avivak-fowl pox”, by the Russian company Avivak NPP (Silva, 2009).

The “AviPro Pox” vaccine is made from chicken embryos infected with bird fowl pox viruses, the “HP-B” strain. The “FPC” strain is used in the production of the German vaccine “AviPro AE+Pox” by the Lohman Animal Health Gmb @Ko KG company.

For the vaccine “Nobilis AE+Pox” the company MSD Animal Health, or Intervet (Holland), uses the “Gibbs” strain. This strain was used also for the “Pigeon Pox” and “AVA+Pox+CE” vaccines (Silva, 2009).

The embryo anti-bird fowl pox vaccine “Avivak Ospa” is made from a live attenuated strain of fowl pox virus, “K” strain. The "Cutter" strain is widely used in the production of the associated vaccines "Gallivac AE+FP" by the Merial company. The “Hitchner”, “Calnek” and “Cutter” strains are used in the production of “Pox blen” associated vaccines. The “Cutter” Strain is also used by the Ceva Sante Animale company in the production of the associated vaccines “Cevac FP+L”,

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Sensitivity of Primary Tripsinized Cell Systems EYQ and FEC to the Fowl Pox Virus 55

“Cevac Poximune”, “Cevac Poximune® AE”, “Cevac Vectormune FP+MG”, “Cevac Vectormune FP+MG+AE”, “Cevac Vectormune® FP+N+A” and “Cevac Vectormune® FP+LT+AE”. The Lohmann Animal Health INT company uses “P” strains for its “AviPro Pigeon Pox P” vaccine. For the “AviPro Pigeon Pox C” vaccine, it uses “C” strain. The “Chicken PV” strain is used for the vaccines “AviPro Pox Ceo”, “AviPro Pox TC”, “AviPro Pox” and “AviPro AE+Pox+TC”. Russian anti-bird fowl pox vaccines include the strains "K", "Ospovak", "27-ASh" and "ND" (Meseko, 2012; Yusifova, 2016; Yusifova, 2015). So, researchers currently use mainly local strains to improve their vaccines. This is important for increased effectiveness of vaccination against fowl pox.

The study in this article is aimed at a comparative analysis of the “Baku” strain of avian pox virus adapted to the culture of quail embryo cells and chicken fibroblast cell culture to obtain a highly pathogenic antigen.

To this end we conducted successive passages of fowl pox virus on primary cell systems to preserve and increase its antigenic properties.

Materials and methods

Cell cultures were prepared by the standard trypsinization method. The sensitivity of the cell culture was determined by infecting it with bird fowl pox virus in dilutions of up to 10– 4. Changes in cell culture were detected by microscopy (Syurin, 1956). During cultivation, the timing of the onset and the nature of the cytopathic effect were determined. The titer was determined by the Reed and Mench method, (EID50/ml), and in the RHA reaction, the virus titer was respectively expressed in HAU50/0,5мл. (Yusifova, 2016; Yusifova, 2015).

Results and discussion

We performed 25 passages of fowl pox virus on cell cultures of Japanese quail embryos and chicken fibroblasts. The biological properties at the time of adsorption of fowl pox viruses in the primary cell systems of Japanese quail embryos and chicken fibroblasts were studied, including the optimal conditions for their reproduction in the systems studied. The destruction of the primary cell culture under the influence of fowl pox virus was observed by microscopy.

Preliminary studies have shown that the cytopathic phenomenon in cell cultures of chicken fibroblast embryos can occur in the first stages of fowl pox virus infection. So, the cytopathic effect in the culture of chicken fibroblast embryo cells was observed in passage 2-3, in the culture of Japanese quail embryo cells in passage 10

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56 Yusifova

on day 5 after infection. In both cell cultures, cell destruction was observed in the first 48 hours, the integrity of the monolayer was not violated. Monolayer destruction was observed in the next 72 hours, and evaluated «+ +» and «+ + +». The cytopathic effect of viruses is equally evident in chicken embryo cell culture and in Japan quail cell culture. But the onset time of the cytopathic effect in the cell cultures was not the same. The cytopathic effect of the fowl pox virus “Baku” strain in the culture of chicken embryo cells was observed 96 hours after infection, but in the culture of quail embryo cells 144 hours after infection.

Conclusion

The results of the studies showed that the fowl pox virus "Baku" strain adapted to both cell systems. We observed an increase in the titer of fowl pox virus in both the culture of Japanese quail embryo cells and in the culture of chicken fibroblast embryos. In both cultures, the virus titer was 256 -512 ГАЕ/0,5ml. It was found that the culture of Japanese quail embryo cells is a more effective tissue culture for vaccine production than chicken embryo cell culture because of its simplicity, economy, absence of extraneous contaminants and its stable biological properties.

It was established that a cell culture of Japanese quail embryos is a promising system for creating highly immunogenic specific prophylaxis against avian fowl pox.

References Cherkezova, T.V. (2003). Materials of the All-Union Scientific Conference “Formation of

immunity to fowl pox in young chickens” p.11- 12, Moscow. Elizbarashvili, E.I. (1987). Features of the cultivation of fowl pox viruses in birds in a

growing and transplanted tissue culture", p.13-15, Moscow. Meseko, C.A., Shittu I.A., & Akinyede O. (2012). Seroprevalence of Fowl Pox Antibody

in Indigenous Chickens in Jos North and South Council Areas of Plateau State, Nigeria: Implication for Vector Vaccine. p.4.

Patrushev, F.M. (1967). The study of some biological properties of fowl pox viruses in birds." Abstract, Perm, p.10-14.

Silva, P.S., Batinga, T.B., & Sales, T.S. (2009). Fowl pox: identification and adoption of prophylactic measures in backyard chickens in Bahia, Brazil; Rev. Bras. Campinas Cienc. Avic. vol.11 no.2, р.4.

Sukhorukov, O.A. (2012). Physiological characteristics of quail and ways of correcting the functional state and production indicators, Tomsk Agricultural Institute - Branch of FSEI HPE Novosibirsk State Agrarian University, Russia, p.3-5.

Syurin, V.N. (1956). Belousova R.V., Fomina N.V. Veterinary Virology.1956. Tarasenko, E.V. (2003). Comparative immunobiological characteristics of vaccine strains

of fowl pox viruses of birds adapted to cell culture», p.4-7, Moscow. Yusifova, K.Y. (2016). Optimal Method of Influenza Virus Infection in the Cell. A: The

"agrarian". № 5, p.25-26, Moscow.

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Sensitivity of Primary Tripsinized Cell Systems EYQ and FEC to the Fowl Pox Virus 57

Yusifova, K.Y. (2015). Biological characteristics of avian pox viruses in cell culture. Proceedings of the II International Conference of the Young Scientists' Council of the Academy of Agrarian Sciences of the MAA. p.149, Baku.

Zenov, N.I. 2010. Quail embryos - the basis for the production of virus vaccines against Marek’s disease and viral diseases in children - measles and mumps, p.2-4.

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Khazar Journal of Science and Technology Volume 3 №1 2019, 58-63 © Khazar University Press 2019 DOI: 10.5782/2520-6133.2019.3.1.58

58

Diurnal Temperature-Related Dynamics of Glutathione Reductase Activity in Wheat Genotypes

Under Drought

Lale Aydinli, Durna Aliyeva

Institute of Molecular Biology and Biotechnologies of ANAS, Laboratory of Bioadaptation and Laboratory of Cell membrane systems

* Corresponding author: [email protected]

Abstract

The strategically important wheat plant is the most cultivated of cereals. Drought is one of the main factors adversely affecting its productivity and quality of grain. Components of the ascorbate-glutathione cycle play an important role in the antioxidant defence system. The diurnal dynamics of temperature-related glutathione reductase activity (GR), one of the main components of the antioxidant defence system, were studied in durum (Barakatli 95, Garagylchyg 2) and bread (Gobustan, Tale 38) wheat genotypes of contrasting tolerance, when exposed to sustained soil drought. Glutathione reductase is highly sensitive to glutathione. Leaf samples were taken at the end of the wax ripening phase at three-hourly intervals (800, 1100, 1400, 1700), frozen in liquid N2, and kept at -800C. The experiments indicated that GR activity increased in stressed, tolerant, (Barakatli 95, Gobustan) and decreased in stressed sensitive varieties (Garagylchyg 2, Tale 38) compared with watered variants. In the samples taken at 1100, enzyme activity increased in all the genotypes, compared with the control. However, in the Gobustan genotype the activity of the enzyme remained almost constant. At the highest temperatures (1400) GR activity decreased by almost 50% both in durum and bread wheat varieties compared with the control. At 1700 GR activity increased in durum wheat genotypes and remained at a low level in bread wheat genotypes. Thus, durum wheat genotypes have a stronger defence system against unfavourable environmental conditions compared with bread wheat genotypes.

Keywords: Triticum aestivum L, Triticum durum Desf., drought, ascorbate glutathione cycle, glutathione reductase.

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Sensitivity of Primary Tripsinized Cell Systems EYQ and FEC to the Fowl Pox Virus 59

Introduction

Strategically important, wheat is one of the world’s most cultivated cereals (Cossani et al., 2012), but its productivity and grain yield are affected negatively by drought (Luo et al., 2009). One of the non-specific responses to drought is oxidative stress accompanied by the intensification of ROS synthesis, which disturbs normal cellular function (Farooq et al., 2009). Hydrogen peroxide, used as a marker of degree of stress, is a stable form of ROS (Rhee et al., 2010). The antioxidant defence system, especially components of the ascorbate-glutathione cycle, implements H2O2 detoxification in plant cells exposed to the effect of stressors (Farooq et al., 2009; Noctor and Foyer, 1998). According to the results of experiments performed on various agricultural species, plant tolerance of stressors - heavy metal pollutants (Smirnoff, 2000) , radiation (Horemans et al., 2000), drought (Esfandiari et al., 2008) and salinity (Joseph and Jini 2011) - depends on the quantities of ascorbic acid and glutathione. Therefore, a determination of antioxidant enzyme activity may be considered an effective method of screening plant drought tolerance. Currently, components of ascorbate-glutathione are the main targets in developing stress-tolerant tansgen plants (Kang et al., 2013). The main purpose of the research presented was to study the temperature-related dynamics of glutathione reductase, one of the main components of the ascorbate-glutathione cycle, in durum and bread wheat genotypes of contrasting drought tolerance.

Materials and methods

Durum (Barakatli 95, Garagylchyg 2) and bread (Gobustan, Tale 38) wheat genotypes of contrasting productivity and drought tolerance, regionalized in Azerbaijan and collected in the genefund of the Research Institute of Crop Husbandry, were used as objects of the research. Leaf samples were taken at the end of the wax ripening phase at three-hourly intervals (800, 1100, 1400, 1700), frozen in liquid N2 and kept at -800C.

MDA quantification

The intensity of lipid peroxidation in plants was assessed on the basis of MDA content in drought-exposed leaf samples. MDA content was measured by spectrophotometry at 532 and 600 nm, based on the reaction with tiabarbituric acid (Heath and Packer, 1968) and calculated by the formula: A (mM/g fresh biomass) = (D532-D600)/46.5

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60 Aydinli

Enzyme extraction

Leaf samples of 0.5g were ground in liquid nitrogen, homogenized in 100 mM Na-phosphate buffer (pH 7.8) containing 1 mM EDTA-Na, 2 mM FMSF, 1% PVP, 0.1% Triton X-100, and centrifuged at 40C, 15000g, for 20 min. The supernatant obtained was used in the analysis of GR.

Glutathione reductase (GR, EC 1.6.4.2) activity assay

Glutathione reductase activity was determined by spectrophotometry at 340 nm, in the presence of the oxidized form of glutathione (GSSH) based on the oxidation of NADPH for 3 min. A Glutathione Reductase Assay Kit (Sigma-Aldrich) was used for this purpose. Enzyme activity was expressed as u/mg protein min, and the coefficient of molar extinction ε=6.2 mM-1cm1.

Total protein assay

Total protein was determined using the Bradford protein assay (Bradford, 1976). A series of Bovine Serum Albumin (BSA) standards was used in the construction of the calibration curve.

Results and discussion

MDA content was found to increase during the day with rising temperatures in both drought-tolerant and -sensitive varieties exposed to drought, compared with watered varieties, and remained at a high level despite the relative decrease in temperature towards the end of the day (1700).

Table. Time course of malondialdehyde content in leaves of wheat genotypes in conditions of control and drought

Genotypes

MDA, (mM/g fresh biomass)

800 1100 1400 1700 Barakatli 95 1.6±0.08 3.4±0.17 3.9±0.2 3.6±0.18 Garagylchyg 2 2.2±0.11 4.1±0.21 4.9±0.25 3.8±0.19 Gobustan 3.4±0.17 3.7±0.19 5.0±0.25 5.0±0.25 Tale 38 3.4±0.17 4.9±0.25 5.6±0.28 3.6±0.18

At 1400 MDA content was at its highest levels in both durum and bread wheat varieties. It remained at a high level in tolerant genotypes (Barakatli 95, Gobustan) at 1700, when the temperature declined, but underwent a relative decrease in sensitive varieties (Garagylchyg 2, Tale 38).

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Sensitivity of Primary Tripsinized Cell Systems EYQ and FEC to the Fowl Pox Virus 61

A direct relation between plant drought tolerance and antioxidant system induction is confirmed by numerous facts. Glutathione reductase activity and other parameters characterizing antioxidant status were different in plant varieties with contrasting tolerance. Glutathione reductase is highly sensitive to glutathione and, at the same time, it can catalyse the reduction of other compounds with disulfide bonds. This enzyme detoxifies H2O2 by reducing the oxidized form of glutathione using NADPH: GSSG + NADPH + H+→2GSH + NADP+. Analysis of leaf samples taken at 800, indicated an increase in GR activity in tolerant (Barakatli 95, Gobustan) and a decrease in sensitive genotypes (Garagylchyg 2, Tale 38) exposed to drought (Figure 1). In samples taken at 1100, enzyme activity increased in all genotypes, compared with the control, except the Gobustan genotype, in which activity remained almost constant. At 1400 enzyme activity decreased by almost 50% in both durum and bread wheat varieties, compared with the control. At 1700, due to the relative decline in temperature, GR activity increased in durum wheat genotypes, compared with the control, but remained at a low level in the bread wheat genotypes. This confirms that durum wheat genotypes have a stronger defence mechanism against adverse environmental conditions than bread wheat genotypes.

Fig. 1. Time course of total GR activity in leaves of wheat genotypes grown under conditions of control and drought stress: A) – Barakatli 95, B) -

Garagylchyg 2, C) – Gobustan, D) – Tale 38

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62 Aydinli

Lascano et al. (2001) reported an increase in glutathione reductase (GR) activity in tolerant wheat genotypes and a greater decline in reduced glutathione (GSH), ascorbate content and less oxidative damage than in susceptible genotypes.

Conclusion

According to the results obtained, drought changes the balance between the synthesis of reactive oxygen species and the antioxidant defence system in wheat genotypes exposed to drought. Components of the ascorbate-glutathione cycle play the major role in the detoxification of H2O2 formed in wheat genotypes due to the effect of drought. Study of the diurnal temperature-related dynamics of glutathione reductase activity demonstrated that the defence system in hexaploid wheat genotypes is weaker than that in tetraploid wheat genotypes.

Acknowledgment

This work was supported by the Science Development Foundation under the President of the Republic of Azerbaijan - Grant № EİF-KETPL-2-2015-1(25) - 56/35/3.

References Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72:248-254.

Cossani, C. M., & Reynolds, M. P. (2012). Physiological traits for improving heat tolerance in wheat. Plant physiology. 160(4):1710-1718.

Esfandiari, E., Shakiba, M. R., Mahboob, S. A., Alyari, H., & Shahabivand, S. (2008). The effect of water stress on antioxidant 427 content, protective enzyme activity, proline content and lipid 428 peroxidation in wheat seedling. Pak. J. Biol. Sci. 11 (15), 1916– 429 1922.

Farooq, M., Wahid, A., Kobayashi, N., Fujita, D., & Basra, S. M. A. (2009). Plant drought stress: effects, mechanisms and management. Agron. Sustain.Dev., 29(1), 185–212.

Heath, R. L., & Packer, L. (1968). Photoperoxidation in isolated chloroplast. I. Kinetics and stoichiometry of fatty acid peroxidation. – Arch. Physiol. Biochem. 125: 189-198, 1968.

Horemans, N., Foyer, C. H., Potters, G., & Asard, H. (2000). Ascorbate function and associated transport systems in plants. Plant Physiology and Biochemistry 38: 531–540.

Joseph, B., & Jini, D. (2011). Development of Salt Stress-Tolerant Plants by Gene Manipulation of Antioxidant Enzymes. Asian Journal of Agricultural Research, 5, 17-27.

Kang, N. Y., Cho, C., & Kim, J. (2013). Inducible expression of Arabidopsis response regulator 22 (ARR22), a type-C ARR, in transgenic Arabidopsis enhances drought and freezing tolerance. PLoS One. 8(11): e79248.

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Sensitivity of Primary Tripsinized Cell Systems EYQ and FEC to the Fowl Pox Virus 63

Lascano, H. R., Antonicelli, G. E., Luna, C. M., Melchiorre, M. N., Gomez, L. D., Racca, R. W., Trippi, V. S., Casano, L. M. (2001). Antioxidant system response of different wheat cultivars under drought: Field and in vitro studies. Aust J Plant Physiol, 28:1095-1102.

Luo, R., Wei, H., Ye, L., Wang, K., Chen, F., Luo, L., Liu, L., Li, Y., Crabbe, M. J., Jin, L., Li, Y., & Zhong, Y. (2009). Photosynthetic metabolism of C3 plants shows highly cooperative regulation under changing environments: a systems biological analysis. Proceedings of the National Academy of Sciences. 106(3):847-852.

Noctor, G., & Foyer, C. H. (1998). Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol., 49(1), 249–279.

Rhee, S., Chang, T. S., Jeong, W., & Kang, D. (2010). Methods for detection and measurement of hydrogen peroxide inside and outside of cell. Mol. Cells, 29(6), 539–549.

Smirnoff, N. (2000). Ascorbic acid: metabolism and functions of a multifaceted molecule. Current Opinion in Plant Biology 3: 229–235.

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Khazar Journal of Science and Technology Volume 3 №1 2019, 64-83 © Khazar University Press 2019 DOI: 10.5782/2520-6133.2019.3.1.64

64

Knowledge, Attitudes and Perceptions (KAP) on Point of Sale (PoS) Advertising and Promotion of Tobacco Bans Among Parents: A Cross-Sectional

Study in Nur-Sultan, Kazakhstan

Arailym Nurmasheva Nazarbayev University School of Medicine, Nur-Sultan, Kazakhstan

Abstract

In Kazakhstan in 2004, the percent out of all deaths that were due to smoking was 24%, twice the worldwide percent (12.0%) and about 50% higher than the percentages in the Russian Federation and Eurasia as a whole (both 16%). Based on the Global Youth Tobacco Survey (GYTS) among those who visited a point of sale (where products are purchased) in the past 30 days, overall 13.6% (14.3%=boys, 13.0%=girls) noticed tobacco advertisements or promotions. Studies have shown that exposure to Point of Sale (PoS) advertising and promotion of tobacco products is associated with increased youth smoking initiation. Nonetheless, to the best of our knowledge, there are no studies that examine the perceptions and attitudes of the parents of these youth towards PoS Tobacco Advertising Promotion and Sponsorship (TAPS) ban. The study objectives include: (1) determining parents’ perceptions of PoS advertising and promotion of tobacco bans; (2) assessing parents’ and experts’ knowledge on PoS TAPS ban definitions and regulations.

Introduction

Tobacco use is the most preventable cause of death universally, causing over 100 million deaths worldwide in the 20th century (WHO, 2017). In Kazakhstan in 2004, the percent out of all deaths that were due to smoking was 24%, twice the worldwide percentage (12.0%) and about 50% higher than the percentages in the Russian Federation and Eurasia as a whole (both 16%) (GATS Country Report, 2014). In January 2006, Kazakhstan ratified the World Health Organization (WHO) Framework Convention on Tobacco Control (FCTC). Thus, like other party countries, have agreed to establish, implement and assess effective tobacco control programs, to measure progress in reaching the goals of the WHO FCTC. Moreover, through the implementation of FCTC Kazakhstan has obliged itself to protect people

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Knowledge, Attitudes and Perceptions (KAP) on Point of Sale (PoS) Advertising and Promotion of Tobacco Bans Among Parents:... 65

from tobacco smoke (The Law of RK on ratification of WHO FCTC, 2006). Later on, in 2009 the Republic of Kazakhstan (RK) adopted a code on the health of the people and the health care system, which contained a comprehensive statement on tobacco control (Article 159) (The Code “On health of people and health care system” 2009).

The Article 13 of the WHO FCTC prohibits tobacco advertising, promotion and sponsorship (TAPS), including with point of sale (PoS) displays. A point of sale concerns any location where tobacco products are displayed, advertised and purchased. PoS consists of not only the final point of purchase (i.e., the register) but also advertising at retail locations (both indoor and outdoor), product display, and price (i.e., tobacco branded cash register, colorful PoS cigar display next to candy) (Lovato et al., 2007). The Article 13 of FCTC recommends a complete advertising ban, within five years of entry into force of FCTC for each party. Currently, of all (181) parties, 72% (131) disclosed having a comprehensive ban on all TAPS. Nonetheless, interpretation of comprehensive ban varies across countries, and do not always include all of the specific standards outlined in the implementation guidelines. In 2018, 60% of all parties included PoS display bans in their extensive ban on TAPS (WHO, 2018).

Point of sale (PoS) promotion is a variation of activities in the retail setting to expand sales of tobacco products. This could include, but not be limited to tobacco advertising signs, branded product giveaways, branded functional items, price discounts and the display of the tobacco products themselves. PoS promotion uses targeted marketing to increase its power by dividing customers into groups and adjusting advertising to attract them. This market segmentation is established upon aspects like consumer or potential consumer demographics, product use, lifestyle or location (Grier and Kumanyika, 2010).

Even though practices differ by region, the tobacco industry is commonly responsible for retail tobacco promotion, providing “power walls” (rows of packaged tobacco product in excessive quantities generally visible in the checkout area) to tobacco vendors and helping with in-store advertising placement (Henriksen, 2012). In India and the United States, tobacco manufacturers pay store owners to demonstrate advertisements (Chaudhry et al., 2007 and Paynter, 2009). In Kazakhstan too some of the store owners confessed about being paid by tobacco manufacturers to display advertisements or install power walls. Tobacco companies persist on targeting areas with a high proportion of youth by selling tobacco products in point of sales near schools and placing tobacco products near candies. Research shows that tobacco advertising both encourages initiation among youth and obstructs cessation in current tobacco users (National Cancer Institute, 2008).

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66 Nurmasheva

Pack image advertises tobacco branding effectively, children are the most responsive when it comes to tobacco advertising. In both Canada and the United States, more PoS promotion in stores was correlated with either a higher proportion of underage shoppers or proximity to an elementary or secondary school (Henriksen et al., 2004; Schooler et al., 1996, and Cohen et al., 2008). TAPS exposure is associated with higher smoking prevalence rates, especially with initiation and continuation of smoking among adolescents (Saffer and Chaloupka, 2000 and Yang T et al., 2012). Furthermore, exposure to TAPS results in around third youth experimentation with tobacco (Emery et al., 1999). According to global Global Youth Tobacco Survey (GYTS) in 2002, 78% of students aged 13-15 years report being exposed regularly to some form of TAPS (GYTS, 2002). In Kazakhstan GYTS in 2009 showed that 36.3% ever smokers-initiated smoking before age ten (Boys = 42.4%, Girls = 26.9%) (GYTS, 2009). Latest GYTS in Kazakhstan, which was carried out in 2014 indicates that 2.0% percent of boys, 1.3% of girls, overall 1.7% of students aged 13-15 years currently smoked cigarettes. Among those who visited a point of sale in the past 30 days, overall 13.6% (14.3%=boys, 13.0%=girls) noticed tobacco advertisements or promotions (GYTS, 2014).

The majority of studies focus on relationship between youth attitudes and exposure to PoS advertising and promotion of tobacco products and smoking initiation. Strong public support is one of the encouragements of the tobacco control advocacy in the world. It is crucial to understand how to raise public support and awareness for banning PoS displays and recognizing the fundamental factors of public support in the general population. To the best of our knowledge, this is the first study that examines parent’s perceptions towards PoS TAPS ban. The present study will help in better understanding perceptions and attitudes of parents regarding PoS TAPS bans. Also, will provide new information on how to support tobacco control advocacy and strengthen government’s obligations to implement ban on PoS TAPS. The study has following objectives: (1) determining parents’ attitudes and perceptions of PoS advertising and promotion of tobacco bans; (2) assessing parents’ knowledge on PoS TAPS ban definitions and regulations.

Materials and methods

15 public schools (12 are located on the right bank and 3 on the left bank of the Ishim River) in Astana those who participated in Big Tobacco, Tiny Target project in May 2018 were randomly selected for this study; 2 schools refused to participate. Parents of middle and high school students (5-11 grades) were recruited to participate in the study. Inclusion criteria required parents to have a child attending one of the 15

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Knowledge, Attitudes and Perceptions (KAP) on Point of Sale (PoS) Advertising and Promotion of Tobacco Bans Among Parents:... 67

schools described above. Ethics approval for data collection was granted by the Nazarbayev University Institutional Research Ethics Committee.

Data collection

Pre-test of the questionnaire was carried out before start of the data collection. All of the comments were taken into account and some of the questions were changed accordingly. Data were obtained through self-administered and anonymous questionnaires. Demographics part of the questionnaire was taken from the Global Adult Tobacco Survey (GATS) which were reviewed and approved by international experts. Students were asked to give questionnaires to one of their parents and bring them back after completion. The questionnaire took 7-10 minutes to complete and included questions on personal smoking behaviors, knowledge on PoS TAPS terminology, and attitudes towards PoS TAPS ban laws, exposure to tobacco advertising and display of tobacco products at PoS in different countries.

Data preparation

Prior to data analysis, cases missing information on the three main outcomes were excluded from the data set. This resulted in a final sample of 514 parents (Table 1).

Table 1. Demographics of respondents (univariate analysis) Parents (n=514) Mean age 41 years Gender Female 67.7% Male 32.3% Smoking Status Smokers 13%

Non-smokers 75.2% Smokers in the past 11.8%

Education level Low (lower than college/university) 27.5%

High (college/university and higher) 72.5% Income

Low (0-150,000 tg) 50.2%

Middle (150,000-300,000 tg) 37.5%

High (>300,000 tg) 12.3%

Nationality Kazakh 71.7%

Russian 16.1%

Other 12.2%

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Employment Government Employee 34.8% Non-government Employee 27.1% Self-Employed 24.5% No job 13.6%

Study Measures

Dependent variables

Attitudes towards display of tobacco products at PoS in Russia. We assessed each participant’s attitude towards display of tobacco products at PoS in Russia by asking a question: “would you favor or oppose the following types of prohibition of display of tobacco products?” and providing a picture of partial TAPS ban in Russia. Answers were as follows: “favor”, “oppose” and “don’t know”.

Attitudes towards display of tobacco products at PoS in Norway. We assessed each participant’s attitude towards display of tobacco products at PoS in Norway by asking a question: “would you favor or oppose the following types of prohibition of display of tobacco products?” and providing a picture of complete TAPS ban in Norway. Answers were as follows: “favor”, “oppose” and “don’t know”.

Attitudes towards display of tobacco products at PoS in Kazakhstan. We assessed each participant’s attitude towards display of tobacco products at PoS in Kazakhstan by asking a question: “would you favor or oppose the display of tobacco products at points of sale at present time?” and providing a picture of current state (no ban) of TAPS at PoS in Kazakhstan. Answers were as follows: “favor”, “oppose” and “don’t know”.

Independent variables

Smoking status. We assessed participants’ smoking status by asking a question: “What is your smoking status?” The options for the answers were: “I have never smoked”, “I have smoked in the past” and “I am a smoker/ I smoke now”.

Attitudes towards PoS TAPS bans. We assessed participants’ attitudes towards PoS TAPS ban by asking a question: “Are you in favor of Point of Sale (PoS) advertising and promotion of tobacco bans?” The options for the answers were: “yes”, “no” and “don’t know”.

Perceptions on PoS tobacco displays and adolescent motivation to smoke. We assessed whether participants thought that PoS tobacco displays motivated adolescents to smoke by asking a question: “Do you think tobacco point of sale

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displays (at stores, kiosks, shopping malls etc.) motivate young people to smoke?” The options for the answers were: “yes”, “no” and “don’t know”.

Perceptions on PoS tobacco displays as advertisements. We assessed whether or not participants perceived on PoS tobacco displays as advertisements by asking a question: “Do you think PoS displays are advertisements?” The options for the answers were: “yes”, “no” and “don’t know”.

Attitudes towards children’s exposure to tobacco products at PoS. We assessed participants’ attitudes towards children’s exposure to tobacco products at PoS by asking a question: “How do you feel about children being exposed to display of tobacco products at points of sale?” The options for the answers were: “positive”, “negative” and “neutral”.

Concern of exposure level to tobacco products at PoS. We assessed participants’ concern towards their own children’s level of exposure to tobacco products at PoS by asking a question: “How important is the level of exposure to display of tobacco products at the point of sale to your child for you?” The options for the answers were in a likert scale: “not important”, “somewhat important”, “very important” and “essential”.

Statistical Analyses

Frequency counts were computed for the demographic variables of the respondents including sex, age, education level, employment and smoking status, income and nationality. For each outcome variable, data were analyzed to determine the relationship with independent variables. Chi-square analyses were used to test the statistical associations between attitudes towards displays of tobacco products at PoS in three countries and smoking status, attitudes towards PoS TAPS bans, whether or not parents perceived that PoS tobacco product displays motivated youth to smoke, whether or not parents perceived PoS displays of tobacco products as advertising, and how parents perceived the level of importance of exposure to PoS tobacco product displays for influencing youth, and attitudes towards children’s exposure to tobacco products at PoS.

Results

Frequencies and Descriptive Statistics

The response rate was 88.4%. The sample was 67.7% female and 32.3% male with the mean age of 41 (minimum age=25, maximum age=69). 75.2% were never

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smokers, 13% were current smokers and 11.8% were smokers in the past. Education level was categorized into two groups; high level of education included those with completed college/university degree or higher and low level of education covered those with incomplete college/university degree or lower. 72.5% had high level of education and 27.5% had low level of education. Income was categorized into three groups: low income denoting “0” consisted of monthly salaries ranging between 0-150 thousand tenge, middle income denoting “1” consisted of monthly salaries ranging between 150-300 thousand tenge and high income denoting “2” consisted of monthly salaries of 300 thousand tenge and higher. The percentages were 50.2%, 37.5% and 12.3% respectively. The sample was pretty diverse in term of nationalities, but we decided to categorize respondents by the most common nationalities which were Kazakh (71.7%) and Russian (16.1%), other nationalities were grouped as “other” (12.2%). Employment was divided into four groups: government employee (34.8%), non-government employee (27.1%), self-employed (24.5%) and those with no job including housewives, retired, students, unable to work and those who answered, “don’t know” (13.6%).

FCTC 13 knowledge

We tested respondents’ knowledge on WHO FCTC Article 13 by asking if they knew about its existence and providing some background information about its guidelines. 56.7% answered “yes” and 43.3 answered “no”.

Attitudes towards a law prohibiting all advertisements for tobacco products

44.2% of participants would favor, whereas, 36.6% would oppose a law prohibiting all advertisements for tobacco products. 19.2 answered “don’t know”.

Attitudes towards PoS TAPS ban

Respondents were asked whether they are in favor of Point of Sale (PoS) TAPS bans. Answers were as follows: “yes”=77.3%, “no”=22.7%.

Knowledge of terminology

We assessed participants’ knowledge on term/expression “display of tobacco products at points of sale” and results revealed that the majority 62.8% did not know the term, as opposed to 37.2% of those who knew it.

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Attitudes towards a law that would completely prohibit display of tobacco products at points of sale

Almost half (49.8%) of the respondents would favor such law. Percentages split quite evenly between answers “oppose”=24.2% and “don’t know”=26%.

Awareness of tobacco advertising at PoS

We also asked participants whether they saw any advertisements or promotions for tobacco products at points of sale (such as stores, kiosks, etc.) during the past 30 days. 50.6% answered “no”, 36.7% answered “yes” and 12.7% answered that they did not visit any points of sale in the past 30 days.

Perceptions of tobacco PoS display’s impact on young people’s motivation to smoke

67.4% of parents thought that tobacco PoS displays motivate adolescents to smoke. Whereas 17.5% of parents thought otherwise and 15.1% answered “don’t know”.

Perceptions of tobacco PoS display as advertisements

Likewise, we assessed whether respondents perceive tobacco PoS display as advertisements. The distribution of answers was almost equal: 50.3% perceived PoS displays as advertisements, and 49.7% did not.

Attitudes towards students’ exposure to PoS tobacco displays

74.9% of parents feel negatively (do not want youth to be exposed) about students being exposed to PoS tobacco displays. 21.3% feel neutrally (do not care about exposure) and 3.8% feel positively (want youth to be exposed).

Concern of exposure level to PoS tobacco displays

In addition, we evaluated how important is the level of exposure to display of tobacco products at PoS to their own child is for the parents. The majority (54.4%) of the parents indicated that it was “very important” (they are very concerned about their child being exposed to tobacco products at PoS) to them. Following with “essential” (they are extremely concerned about their child being exposed to tobacco products at PoS) (25.3%), “not important” (they are not concerned about their child being exposed to tobacco products at PoS) (10.3%) and “somewhat important” (they are somewhat concerned about their child being exposed to tobacco products at PoS) (10%).

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Attitudes towards display of tobacco products at PoS in Russia, Norway and Kazakhstan (Table 2).

Table 2. The following table shows results for three countries for parents Display Russia Display Norway Display Kazakhstan Favor 165 (32.1%) 184 (35.8%) 53 (10.3%) Oppose 206 (40.1%) 171 (33.3%) 351 (68.3%) Don’t know 143 (27.8%) 159 (30.9%) 110 (21.4%)

Moreover, we carried out Pearson’s chi square tests to further examine outcome variables such as display of tobacco products at PoS in three countries and whether they differ by smoking status, attitudes towards PoS TAPS ban, whether or not they think that PoS tobacco displays motivate youth to smoke, whether or not they perceive PoS tobacco displays as advertisements, attitudes towards children’s exposure to tobacco products at PoS, and concern of level of exposure to tobacco products at PoS.

Bivariate analyses

Attitudes towards display of tobacco products at PoS in Russia and independent variables

Most of the non-smokers (42.8%) and smokers in the past (41.7%) opposed type of tobacco products display in Russia, while the majority of current smokers answered “don’t know” (40.9%).

Attitudes towards PoS TAPS ban were consistent with the answers given to type of tobacco products display: majority (47.4%) of those who favored Russia’s type of display also favored a law that completely prohibits display of tobacco products at points of sale; the results are the same for those who opposed (68.9%) and answered “don’t know” (51.9%).

Regarding perceptions on PoS tobacco products displays and adolescent motivation to smoke, only group of parents who answered “don’t know” (44.74%) were consistent with their answers related to whether they think that PoS tobacco displays motivate students to smoke. Of those who answered negatively, equal amount (35.2%) of people favored and was not sure about Russia’s type of tobacco display, whereas 29.6% opposed. Most of parents (42.8%) who think that PoS tobacco displays motivate young people to smoke, opposed Russia’s type of tobacco display.

Regardless of whether or not respondents perceived PoS tobacco displays as advertising, many of them opposed Russia’s type of tobacco display. However, it is worth noting that of those who perceived PoS tobacco displays as advertising, got

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split almost equally between favoring (38.8%) and opposing (39.1%) display of tobacco products at PoS in Russia.

In addition, participants’ attitudes towards children’s exposure to tobacco products at PoS were consistent with their opinions on Russia’s type of tobacco display. Most (47.2%) of those who feel negative about children being exposed to display of tobacco products at PoS opposed type of tobacco display in Russia. Most (50.9%) of those who feel neutral about children being exposed to display of tobacco products at PoS answered “don’t know” regarding type of tobacco display in Russia. Most (68.4%) of those who feel positive about children being exposed to display of tobacco products at PoS favored type of tobacco display in Russia.

Moreover, concern of level of exposure to tobacco products at PoS for their own child was distributed between different opinions on Russia’s type of tobacco display as follows: for those to whom level of exposure was “not important”, more than half (51.9%) expressed their ambiguity towards type of display in Russia; likewise, for those to whom level of exposure was “somewhat important”, majority (39.2%) expressed their ambiguity towards type of display in Russia; for those to whom level of exposure was “very important”, most of the respondents (44.7%) opposed; for those to whom level of exposure was “essential”, majority of the participants (38.2%) favored type of tobacco display in Russia.

Attitudes towards display of tobacco products at PoS in Norway and independent variables

The majority of non-smokers (35.8%) opposed type of tobacco products display in Norway. Most of the current smokers (42.4%) answered “don’t know” and 45% of past smokers favored Norway’s type of tobacco display.

Likewise the case with Russia’s type of tobacco display, attitudes towards PoS TAPS ban were consistent with the answers given to type of tobacco display in Norway: the majority (50.8%) of those who favored Norway’s type of tobacco display also favored a law that completely prohibits display of tobacco products at points of sale; the results are the same for those who opposed (60.7%) and answered “don’t know” (50.4%).

Similarly, perceptions on PoS tobacco displays and adolescent motivation to smoke were analogous to opinions on type of display, except those who answered negatively: most of (50%) respondents who were ambiguous about their perceptions were also ambiguous about whether they would oppose or favor the Norway’s type of tobacco display. Most of those who answered “yes” (39.6%) and “no” (39.8%)

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in regard to perceptions on PoS tobacco displays and adolescent motivation to smoke favored type of tobacco display in Norway.

In addition, most of those (44.6%) who perceived PoS tobacco displays as advertising favored tobacco display type of Norway. Most of those (37%) who did not perceive PoS tobacco displays as advertisements opposed tobacco display type of Norway.

Regarding participants’ attitudes towards children’s exposure to tobacco products at PoS, results were consistent with their opinions on Norway’s type of tobacco display. Most (38.1%) of those who feel negative about children being exposed to display of tobacco products at PoS opposed type of tobacco display in Norway. Most (45.4%) of those who feel neutral about children being exposed to display of tobacco products at PoS answered “don’t know” regarding the type of tobacco display in Norway. Most (63.2%) of those who feel positive about children being exposed to display of tobacco products at PoS favored type of tobacco display in Norway.

Furthermore, following is the distribution of concern of level of exposure to tobacco products at PoS for their own child between different opinions on Norway’s type of tobacco display : for those to whom level of exposure was “not important”, 44.2 % showed their ambiguity towards type of display in Norway; for those to whom level of exposure was “somewhat important”, majority (39.2%) expressed their ambiguity towards type of tobacco display in Norway; for those to whom level of exposure was “very important”, most of the respondents (38.3%) opposed; for those to whom level of exposure was “essential”, majority of the participants (42.1%) favored type of tobacco display in Norway.

Attitudes towards display of tobacco products at PoS in Kazakhstan and independent variables

Regardless of their smoking status, majority of the participants, 72.7% of non-smokers, 42.4% of current smokers and 70% of smokers in the past opposed current state of tobacco display at PoS in Kazakhstan.

Attitudes towards PoS TAPS ban were consistent with the answers given to type of tobacco display in Norway only for two groups: majority (80.3%) of those who opposed Kazakhstan’s type of tobacco display would also oppose a law that completely prohibits display of tobacco products at points of sale; most (48.9%) of those who answered “don’t know” would also be ambiguous about display type. 75.3% of those who opposed Kazakhstan’s type of tobacco display favored the law.

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Same as with the smoking status, regardless of their perceptions on PoS tobacco displays and adolescent motivation to smoke, most of the parents, specifically, 56.6% of those who answered “don’t know”, 57.9% of those who answered “no” and 74.3% of those who answered “yes” opposed current state of display of tobacco products in Kazakhstan.

Similarly, regardless of whether they perceive PoS displays as advertising or not, greater part of respondents 71.3% and 65.5%, respectively, opposed type of tobacco display in Kazakhstan.

Respondents’ attitudes towards children’s exposure to tobacco products at PoS were consistent with their opinions on Kazakhstan’s type of tobacco display. Most part (78.4%) of those who feel negative about children being exposed to display of tobacco products at PoS opposed type of tobacco display in Kazakhstan. Many (46.3%) of those who feel neutral about children being exposed to display of tobacco products at PoS answered “don’t know” regarding type of tobacco display in Kazakhstan. The majority (63.2%) of those who feel positive about children being exposed to display of tobacco products at PoS favored type of tobacco display in Kazakhstan.

Furthermore, regardless of their concerns towards importance of exposure level to PoS tobacco displays most of the parents opposed display of tobacco products in Kazakhstan, in particular, of those who answered “not important” 51.9%, of those who answered “somewhat important” 56.9%, of those who answered “very important” 74.9% and of those who answered “essential” 67.2%.

We conducted a one-way ANOVA (Table 3) test to determine if age of the respondents was different for groups with different types of tobacco products’ display in each of the countries.

Table 3. Bivariate analysis –associations between independent variables and outcome variable (display of tobacco products at Point of Sale in Kazakhstan) Smoking status Display of tobacco products at

PoS in KZ Non-smoker Current smoker Smoker in the past oppose 72.70% 42.42% 70.00% Favor 8.14% 15.15% 18.33% don’t know 19.16% 42.42% 11.67%

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Table 3 continued.

Concern of level of exposure to tobacco products at Point of Sale to

their own child Display of tobacco products at PoS in KZ

Not important

Somewhat important Very important Essential

Oppose 51.92% 56.86% 74.91% 67.19% Favor 7.69% 7.84% 11.27% 10.94% Don’t know 40.38 % 35.29% 13.82% 21.88%

Table 3 continued.

In favor of Point of Sale advertising and promotion of

tobacco bans Display of tobacco

products at PoS in KZ No Yes Oppose 60.34% 70.89% Favor 4.31% 12.15% Don’t know 35.34% 16.96%

Table 3 continued.

Law prohibiting all advertisements for tobacco products Display of tobacco products at PoS in KZ Favor Oppose Don't know Oppose 72.69% 76.60% 42.42% Favor 14.54% 5.85% 9.09% Don’t know 12.78% 17.55% 48.48%

Table 3 continued.

Law that prohibits display of tobacco products at Points of

Sale Display of tobacco products at PoS in KZ Favor Oppose Don't know Oppose 75.30% 80.33% 42.75% Favor 12.75% 7.38% 8.40% Don’t know 11.95% 12.30% 48.85%

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Table 3 continued.

Tobacco Point of Sale displays motivate

children to smoke Display of tobacco products at PoS in KZ No Yes Don't know

Oppose 57.95% 74.34% 56.58% Favor 6.82% 13.57% 1.32% Don’t know 35.23% 12.09% 42.11%

Kazakhstan

Participants were classified into three groups: Favor, Oppose and Don’t know. There was a statistically significant difference between groups as determined by one-way ANOVA (F (2,488) = 3.31, P= 0.0357). A Tukey post-hoc test revealed that age was statistically significantly lower in the Oppose group compared to the Don’t know group (-1.85 ± 0.77 years, P= 0.043). However, there were no statistically significant differences between the Favor and Don’t know groups (-2.32 ± 1.18 years, P= 0.123), or the Oppose and Favor groups (0.46 ± 1.04 years, P= 0.896).

Russia

Respondents were classified into three groups: Favor, Oppose and Don’t know. There was a statistically significant difference between groups as determined by one-way ANOVA (F (2,488) = 4.00, P= 0.0189). A Tukey post-hoc test revealed that age was statistically significantly lower in the Oppose group compared to the Don’t know group (-2.06 ± 0.76 years, P= 0.019). However, there were no statistically significant differences between the Favor and Don’t know groups (-0.68 ± 0.80 years, P= 0.671), or the Oppose and Favor groups (-1.38 ± 0.3 years, P= 0.147).

Norway:

Parents were classified into three groups: Favor, Oppose and Don’t know. There was a statistically significant difference between groups as determined by one-way ANOVA (F (2,488) = 3.47, P= 0.0319). A Tukey post-hoc test revealed that age was statistically significantly lower in the Oppose group compared to the Don’t know group (-2.04 ± 0.77 years, P= 0.024). However, there were no statistically significant differences between the Favor and Don’t know groups (-0.96 ± 0.76 years, P= 0.413), or the Oppose and Favor groups (-1.07 ± 0.75 years, P= 0.325).

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Discussion

Present study reveals that majority of parents oppose current state of tobacco products’ display at PoS in Kazakhstan, specifically, 68.3% and favor Norway’s type of tobacco display (35.8%). Norway was the first country to affirm the WHO FCTC in June 2004. After that in the 1st of January 2010, Norway implemented the PoS display ban for tobacco products along with smokeless tobacco (snus) and smoking accessories. Retail outlets can choose how to cover up tobacco products. Commonly, tobacco products are placed in closed containers above or below the counter, in closed drawers or cabinets or behind sliding doors, shutters or similar solutions (Examples in Picture 1a and b). Price lists must contain only neutral price information with no pictures or other types of information. They can be presented next to the cash register or given to the customer upon request. Only one list per cash register is allowed (Ollila, WHO FCTC Convention Secretariat, 2015).

In Kazakhstan there is no such ban. Tobacco products are openly displayed at PoS. To make tobacco products more attractive to young people they are placed on the eye level of children and near candies and toys, stimulating children to see them as not dangerous everyday items (Examples in pictures 2a and b). That is the case across the board in countries with no effective PoS TAPS ban. There was a study among students aged 13-16 years in Scotland, exploring relationship between PoS displays of cigarettes and brand awareness. It revealed that students visiting small shops more frequently and those who recognized cigarette displays had higher brand awareness. In addition, adolescents described PoS tobacco displays as being colorful, eye-catching and potentially attractive to young people (Sluijs et al., 2016).

More than half (56.7%) of respondents knew about WHO FCTC Article 13 and its guidelines. However, most of them (62.8%) did not know the term “display of tobacco products at points of sale”. Nonetheless, 77.3% of participants had positive attitudes towards PoS TAPS bans and 49.8% favored a law that would completely prohibit display of tobacco products at PoS. 67.4% of parents recognized tobacco PoS displays as a motivation for adolescents to smoke. Perceptions of respondents have been proven to be true scientifically. A study conducted in UK shows that among non-susceptible never smokers seeing tobacco PoS displays more often is associated with a higher risk of becoming susceptible to smoking [adjusted relative risk ratio (RRR)=1.74]. Identifying a higher number of brands among non-susceptible never smokers the risk of becoming susceptible to smoking and of becoming a smoker was twice higher (Bogdanovica et al., 2014). Likewise, a study among Nigerian adolescents revealed that cumulative TAPS exposure was significantly associated with increased odds of cigarette use (AOR=1.73) and ever

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cigarette use (AOR=1.29); along with increased susceptibility to cigarette smoking (AOR=1.18) among non-smokers (Chido-Amajuoyi et al., 2017).

This study shows that majority (74.9%) of parents had negative attitudes about students being exposed to PoS tobacco displays. Furthermore, concern of level of exposure to display of tobacco products at the PoS for their own children was assessed. Most (54.4%) of the parents indicated that it was “very important” to them, following with “essential” (25.3%). According to the study in the UK, there was significantly less parental tolerance for child exposure to nicotine/smoking than gambling and alcohol (Hood and Parke, 2015). This indicates that children’s’ exposure to tobacco products and perceptions of that exposure are of high importance to parents.

As stated in the meta-analysis (consisting of 13 studies) on PoS tobacco promotion and youth smoking: children and adolescents more commonly exposed to PoS tobacco promotion have about 1.6 times higher odds of having tried smoking and about 1.3 times higher odds of initiating smoking in the future, compared to those less commonly exposed (Robertson et al., 2015). In accordance with another review (including 19 longitudinal studies) which examined impact of tobacco advertising and promotion on increasing adolescent smoking behaviors, results imply that exposure to tobacco advertising and promotion is linked to the probability that young people will start to smoke. Authors of the review conclude that tobacco advertising and promotion increases adolescents’ possibility smoking initiation based on evidence of a dose-response relationship, homogeneity of findings across different observational studies, strength and specificity of this association, along with theoretical plausibility in regard to effect of advertising (Lovato et al., 2011).

Two experimental studies examined effect of changing the location or visibility of the tobacco power wall in a life sized replica of a convenience store on teenagers’ susceptibility to future cigarette use. According to the study conducted in 2015 hiding the tobacco power wall substantially decreased young people’s susceptibility to future cigarette smoking compared to leaving it exposed (p=0.02). Placing the tobacco power wall on a sidewall away from the cashier had no impact on future cigarette use susceptibility compared to the exposed position (p=0.80) (Shadel et al., 2015). Similarly, more recent study suggests that attention toward the tobacco power wall was found to be importantly related to future smoking susceptibility with p=0.046, while adjusting for baseline cigarette smoking susceptibility and other potential confounders (S.C. Martino et al., 2018).

Furthermore, there have been studies carried out to test the effect of PoS TAPS bans. In July 2012 New Zealand implemented PoS TAPS ban. A study evaluating impact

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of this tobacco control act showed that between 2011 and 2014, smoking experimentation declined from 23% to 17% (AOR=0.73); current smoking rates from 9% to 7% (AOR=0.71), initiation in the last year from 13% to 11% (AOR=0.91). Attempted cigarette purchase in the past 30 days among smokers had fallen from 30% to 26% (AOR=0.77) (Edwards et al., 2016). Another study assessed effect of PoS tobacco display removal on smoking behavior among adolescents in 25 European countries. Results suggest that regardless of gender, enforcement of a PoS display ban was related to 15% decline in the odds of regular smoking (OR=0.85) but was not significantly linked to perceived accessibility of tobacco (OR=0.97) (Van Hurck et al., 2018). Ireland enabled PoS tobacco display ban in July 2009. Following the implementation, a study was conducted to assess the short-term effects of the ban. It showed that display recall declined substantially for adults (from 49% to 22%; p<0.001), even more for adolescents (from 81% to 22%, p<0.001). After legislation, 14% of adult smokers thought that the law had made it easier to quit, 38% of adolescents thought that the law would make it easier for children not to smoke (Mc Neill et al., 2010).

As it was stated in the WHO MPOWER 2017 report 7 primarily low and middle income countries, specifically, Uganda, Senegal, Nigeria, Afghanistan, and Republic of Moldova introduced an extensive TAPS ban, as well as, at the PoS (WHO, 2017). During the Seventh session of the Conference of the Parties (COP) in 2016 to the WHO FCTC accepted the United Nation’s (UN) 2030 Agenda for Sustainable Development, along with Sustainable Development Goal (SDG) 3, to “ensure healthy lives and promote well-being for all at all ages”. In addition, target 3A of the goal is to “strengthen the implementation of the WHO FCTC in all countries, as appropriate” as a way of reaching SDG 3 by 2030 (UN, 2017). In my opinion, Kazakhstan should follow the lead of countries who have implemented comprehensive PoS TAPS bans in order to reach SDG 3 and guarantee well-being of its nation.

Currently, The RK’s law on advertising of 2003 (as amended in June 2007 by the law on amending legislative proclamations of advertising) is the principal law in the republic controlling the advertising of tobacco products; it provides a general ban on the advertising of tobacco and tobacco products, as well as the forms of sponsorship and promotion. Additionally, the law bans the advertising of goods (works, services) with the elements of a trademark or name that is known as the name of tobacco or tobacco products (The Law of RK, 2007). Nonetheless, according to the Global Adult Tobacco Survey (GATS) Kazakhstan 2014 results 25.7% of adults noticed advertising, sponsorship or sales promotion of cigarettes, whereas 14.0% of adult population noticed the cigarette advertising at PoS, 2.6% on television, 2.8% on

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billboards, and 7% on the Internet. Overall, 83.9% of adults were in favor of a total ban on tobacco products advertising (GATS RK Global Report, 2014).

Strengths and limitations

Present study has a few strengths. To our best knowledge, this is the first study to assess the knowledge, attitudes and perceptions of parents in regard to PoS TAPS ban. Random selection of schools positively effects generalizability of the results.

This study has some limitations that should be kept in mind when clarifying results. First, since questionnaires were distributed to students first, and then through students to parents, there is a possibility that some of the students would have answered the questionnaires themselves or asked others (older siblings/friends) to fill them out. Second, the results from the self-reported questionnaires could have been subject to response bias.

Conclusion

Public support has been one of the determinants for successful tobacco control advocacy in the world. Results of this study show that parents, despite whether they are smokers or not, oppose current state of PoS TAPS in Kazakhstan. The findings of this study will be valuable for developing PoS TAPS ban policies and programs targeted at parents in Kazakhstan. Strong support by parents strengthens the position of the government to regulate PoS TAPS through building public awareness, advocacy with stakeholders and enforcement of law. References Bogdanovica, I., Szatkowski, L., McNeill, A., Spanopoulos, D., & Britton, J. (2014).

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Information to Contributors Khazar Journal of Science and Technology focuses on the results of original research projects in Mathematics, Physics, Chemistry, Earth Science, Agriculture and Life Sciences as well as Engineering and Technology. The journal is published in English. The Editorial Board does not accept articles published or submitted for publication elsewhere. Articles may be submitted by email. Please submit your manuscript to the following addresses: [email protected] and [email protected] Preparation of Manuscript Manuscripts that are not prepared using the template will not be considered for publication. Manuscript Types Original research articles, review articles, research notes/short communications, case studies/case reports, and letters to the editor are welcome. The editor-in-chief can change the manuscript type after manuscript submission. • Research Articles A research article reports the results of original research and assesses its contribution to the body of knowledge in a given area with the relevant data and findings in an orderly, logical manner. • Review Articles A review article is written to summarize the recent developments, improvements, discoveries, and ideas in various subjects. Review articles should present an unbiased summary of the current understanding of the topic. Review articles should cover subjects that fall within the scope of the journal and are of active, current interest. Review articles should be no longer than 50 pages, should have an abstract of 300 words at most, should contain a limit of 120 references, and should have no more than 12 figures and tables combined. Principal sections should be numbered consecutively (1. Introduction, 2. Historical background, etc.), and subsections should be numbered 1.1., 1.2., etc. All reviews should contain an introduction section and a conclusion section, with relevant section headings in between. The introduction should explain the importance of the subject, the text should be comprehensive and detailed, and the references should be exhaustive. Review articles should be written with the support of original published studies of the author(s). • Research Notes/Short Communications Research notes/short communications are short papers that present original and significant material for rapid dissemination. A research note/short communication may focus on a particular aspect of a problem or a new finding that is expected to have a significant contribution to science. Research notes/short communications should be a concise but complete description of a limited investigation that will not be included in a later paper. These should be as completely documented, both by reference to the literature and by description of the experimental procedures employed, as a regular paper. Research notes/short communications should be no

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