Issue 2 - The University of Zakho

Transcription

Kurdistan Region - Iraq
Ministry of Higher Education
and Scientific Research
University of Zakho
Journal of
University of Zakho
A - Science
Volume 2
Number 2
December
2014
Journal of University of Zakho
A - Science
Volume 2
Number 2
December
2014
Journal of University of Zakho, Vol. 2(A), No.2, 2014
Contents
- VASORELAXANT AND ANTISPASMODIC EFFECTS OF SOME POLYPHENOLS
AND GLYCOSIDE IN RAT SMOOTH MUSCLES
Mudhir S. Shekha ……………………………..………………………………………..…………... 220
- IMPACT OF NUTS CONSUMPTION ON BLOOD PRESSURE, GLYCEMIC
STATUS, LIPID PROFILE AND TOTAL PROTEIN IN HEALTHY HUMAN
VOLUNTEERS
Suad Y. AL-Kass, Omar A. M. Al-Habib and Kajeen H. Jasim……………………………………228
- STUDIES ON SOME ENZYME ACTIVITIES IN LAMINATED AND GERMINAL
LAYERS OF HYDATID CYSTS ISOLATED FROM DIFFERENT INTERMEDIATE
HOSTS IN ZAKHO, DUHOK PROVINCE, KURDISTAN REGION OF IRAQ.
Wijdan M.S. Mero and Araz R.I. AL Bosely ……………………………………………………… 239
- HISTOPATHOLOGICAL STUDY OF THE EFFECT OF CISPLATIN ON THE
OVARIES OF FEMALES' ALBINO MICE
Intissar Numman Waheed, Aveen Abdullah Mohammed Ameen, Unce Jasib Jasim and Bakhtyar
Nader Ali ..............................................................................................................................................245
- NEW RECORDS OF FUNGI ON WHEAT GRAINS FROM IRAQ
Samir Khalaf Abdullah1 and Halben Ismat Mohammad Atroshi2………………………………….. 256
- PATHOGENIC MICROORGANISMS ASSOCIATED WITH DIARRHEA IN
INFANTS AND CHILDREN IN DUHOK PROVINCE, KURDISTAN REGION /
IRAQ”
Awaz H. H. Badry, Ahmed Y. Jameel, Wijdan M. S. Mero………………………………………...266
- HISTOLOGICAL CHANGES IN SOME ORGANS OF THE FEMALE RATS
INFECTED WITH TOXOPLASMA GONDII PARASITE ISOLATED FROM EMBRYO
OF ABORTED SHEEP
Liqaa H. Al-Delami, Buthaina Hatim. Al-Sabawi, Firas M.Basheer, Hafidh I. Al-Sadi…………...276
- INCIDENCE OF HUMAN SCABIES IN DUHOK PROVINCE, KURDISTAN
REGION/ IRAQ
Wijdan M.S. Mero and Haliz Khalid Hassan………………………………………………………. 285
- MULTILOCUS SEQUENCE TYPING OF KLEBSIELLA PNEUMONIAE
PRODUCING EXTENDED SPECTRUM Β-LACTAMASES ISOLATED FROM
KURDISTAN REGION, IRAQ.
Haval Mohammed Khalid, Jaladet M.S Jubrael and Samira Younis Yousif ……………………… 293
Journal of University of Zakho, Vol. 2(A), No.2, 2014
- DACTYLOGYRUS SCRJABINENSIS (MONOGENEA: DACTYLOGYRIDE): FIRST
OCCURRENCE ON THE GILLS OF CAPOETA TRUTTA FROM IRAQ
Younis Sabir Abdullah and Shamall Mohammad Amin A. Abdullah................................................ 299
- IDENTIFICATION OF POTATO VIRUS Y (PVY) AND ITS ECONOMIC
IMPORTANCE ON POTATO CROP
Nabeel Aziz Kassim, Zulaykha Abdulwahab Abduljalil Nerway and Kurdistan Hassan Yousif .... 304
IN
VITRO
MULTIPLICATION
OF
FRASERI)USINGDIFFERENT CULTURE MEDIA
PHOTINIA
(PHOTINIA
X
Layla Shaaban Mohammed AL-Mizory and Ameena Mohammed Hassan……………………….. 310
- APPLICATION METHOD OF POTASSIUM HUMATE ON GROWTH AND YIELD
OF GREEN ONION (ALLIUM CEPA L.)
Kurdistan Hassan Yousif …………………..…………………………..………………………..… 323
HOMOGENEOUS PHOTOCATALYTIC DEGRADATION
ALIZARIN BLACK USING HYDROGEN PEROXIDE
-
OF
ACID
Haydar A. Mohammad Salim, Sabir Ayob Mohammad Salih and Sherwan M. Simo ……………. 329
- PHOTOCATALYTIC DEGREDATION OF ACID ALIZARIN BLACK USING
POWDER AND NANOPARTICLES OF TITANIUM DIOXIDE
Haydar A. Mohammad Salim, Sherwan M. Simo and Neewar A. Yaseen ………………………... 336
- THE HYDROLOGICAL COMPARISIONS BETWEEN THE CHALK AQUIFER
AND THE HOLDERNESS GLACIAL TILL OF SMALL CATCHWATER DRAIN
CATCHMENT IN HOLDERNESS, ENGLAND.
Ramadhan Haji Sulaiman Zaidky …………………….…………………………………………… 342
- NUMERICAL INVESTIGATION OF PRANDTL NUMBER EFFECTS ON THE
NATURAL CONVECTION HEAT TRANSFER FROM CIRCULAR CYLINDER IN
AN ENCLOSED ENCLOSURE
Omar Mohammed Ali …………………………………………………………………………….... 358
- USING MONTE CARLO SIMULATION TO CALCULATE THE DOSE
REDUCTION AT THE MAZE ENTRANCE OF A RADIOTHERAPY ROOM
Dindar Shamsadin Bari ..…………… ..…….……………………………………………………… 375
- BULK ETCH RATE MEASUREMENT OF CR-39 DETECTOR AT CONSTANT
NORMALITY USING THE DIAMETERS AND THE LENGTHS OF THE TRACKS
OF α-PARTICLES
Hussein A. Ahmed ………………………………………………………………………………...…382
Journal of University of Zakho, Vol. 2(A) , No.2, Pp 220-227, 2014
VASORELAXANT AND ANTISPASMODIC EFFECTS OF SOME
POLYPHENOLS AND GLYCOSIDE IN RAT SMOOTH MUSCLES
Mudhir S. Shekha
Department of Biology, Colleges of Science, University of Salahaddin, Kurdistan Region – Iraq.
(Accepted for publication: December 30, 2014)
Abstract:
Objective: The present study was undertaken to investigate the effect of Luteolin, chlorogenic acid,
amygdalin and tannic acid on vascular smooth muscle contractility.
Materials and Methods: The effect of different concentrations of some polyphenols and glycosides on
contractile responses of isolated aorta, ileum and trachea to Acetylcholine, KCl and phenylephrine (PE)
were evaluated.
Results: The results of the current study indicated that luteolin and chlorogenic acid has potent
relaxant effects on KCl and PE-induced contractions in rat’s aortic ring. Amygdalin and tannic acid
reduced ACh-induced ileum contractions significantly. Conclusions: The inhibitory effect of luteolin and
chlorogenic acid on the contraction induced by PE and KCl may be due to their anti-adrenergic and antihyperpolarizing effect.
Keywords: vasorelaxant, antispasmodic, polyphenol, glycoside, rat, smooth muscles
Introduction
ncreasing evidence indicates that regular
and moderate consumption of polyphenolrich beverages or foods may exert protective
effects on the cardiovascular system (Leblais et
al. 2008). Polyphenols represent a superfamily
of diverse naturally occurring phytochemicals
(Menaa et al. 2014).
Flavonols and flavones are plant-derived
polyphenolic compounds that are commonly
consumed in the diet. Epidemiological studies
indicating that high dietary intake of flavonols
reduces the risk of cardiovascular disease and
subsequest mortality (Graf et al. 2005). Luteolin,
a flavone which is the major component in many
herbal plants, has a variety of pharmacological
effects, including antihypertensive (Loizzo et al.
2007), anti-inflammatory (Chen et al. 2014), and
anti-oxidative (Song & Park 2014).
Recently, basic and clinical investigations
have implied that the consumption of
chlorogenic acid have an anti-hypertensive effect
(Zhao et al. 2012). Chlorogenic, the ester of
caffiec and quinic acida, is a well-known
antioxidant agent. Chlorogenic acid plays a
major role in the protective effect against
ischemia-reperfusion injury (Sato et al. 2011)
and also may be beneficial for the prevention
and treatment of inflammations (Hwang et al.
2014).
Amygdalin (vitamin B17, or Laetrile), is
extracted from Semen Persicae, the seed of
Prunus persica (L.), Batsch and other rosaceous
plants (Chang et al. 2006). Amygdalin is
I
effective at alleviating inflammatory pain and it
can be used as an analgesic with anti-nociceptive
and anti-inflammatory activities (Hwang et al.
2008). Besides the antitumor activity amygdalin
has also been used for the treatment of asthma,
bronchitis, emphysema, leprosy and diabetes
(Baroni et al. 2005) and (Hwang et al. 2008).
Tannic acid, (TA) a naturally occurring plant
polyphenol, is composed of a central glucose
molecule reprivatized at its hydroxyl groups
with one or more galloyl residues (Gülçin et al.
2010). Tannic acid has well-described as an
antimutagenic and antioxidant agent (Andrade et
al. 2005).
However, there is a dearth of information’s
on the effect of polyphenol and glycoside on the
contractility smooth muscle, thus, the present
study was designed to investigate the relaxant
action of selected polyphenol and glycoside on
isolated rat thoracic aorta, ileum and trachea.
MATERIALS AND METHODS
Animals
Adult male albino rats Rattus rattus
weighting (200-300 g) were used for all
experiments, kept in plastic cages and
maintained in animal house of the Department of
Biology, College of Science, University of
Salahaddin. They were kept at 24 Co and
exposed to a photoperiod cycle of 12 hrs light
follow by 12 hrs darkness. The had free access
to water but food withdrawn 24 hr. prior to the
experiments. They were fed standard diet and
tap water.
220
Chemicals and Drugs:
Phenylphrine
chloride,
Acetylcholine
hydrochloride, Luteolin (Roth-Germany), and
potassium chloride (BDH - UK ). Fresh
physiological Krebs and Tyrodes solutions were
prepared daily for tissue experiments
Isolated Aorta Preparation and Experimental
Protocol
The animals were injected intrapretoneally
with heparin (2000 units/ 200 gm) and left for 30
min, to avoid blood clotting and possible
damage of endothelium of the aorta(Fulton et al.
1996). Animals then anaesthetized with
Ketamine (40/kg mg) and Xylazine (10 mg/Kg)
intraperitonealy. The chest cavity was opened,
and after removal of excess tissue and fat, the
isolated aorta was transferred to a beaker
containing Krebs solution aerated with 95% O2
and 5% CO2. The beaker was placed in the water
bath at 37 Co. The aorta cut into rings
approximately 3.5 mm. Only the first four
segments distal to the aortic arch were used. Due
to an apparent difference in magnitude of
contraction resulting from aortic ring after 4th
segment.
The procedure of (Shekha & Al-Habib 2012)
was followed to study the vascular activity of
isolated aorta, ileum and trachea. Briefly, it
includes the carful insertion of two stainless steel
wires into lumen of the aortic rings, then one
wire was anchored to the base of the glass organ
bath (High Tech tissue organ bath Radnoti,
Model 166051 /2EA) and the other wire was
connected to a force transducer (Model
MLT0201/RAD 5 mg-25 gm) coupled to the
transbridge amplifier (Model ML 224, Quad
Bridge Amp) and PowerLab Data Acquisition
System
(Model
ML
870,
PowerLab,
ADinstrument , Sydney, Australia) and computer
running Labchart Pro software (Version 7) was
used for measurement of isometric tension.
The organ bath was filled with 10 ml
oxygenated (95% O2 and 5% CO2) Krebs
solution. The temperature of solution inside the
tissue bath was maintained 37 Co by circulating
water through water jacket from a circulating
water bath set at 37 Co (Thermo-Fisher
Scientific, USA).
Aortic Rings were allowed to equilibrate for
60-90 min at a resting tension of 2 gm with
buffer solution changed every 15 minutes. When
isometric tension had stabilized, concentrationresponse curves (CRCs) for acetylcholine 1X10-9
221
– 10-5 mol/L, submaximal dose of phenylephrine
1X10-6 M and KCl 60 mM were established.
Ileum
Preparation
Protocol
and
Experimental
Rats were anaesthetized, the abdomen was
opened, the caecum was lifted forward and the
ileo-caecal junction was exported. The ileum
was cut at the junction and transferred into a
beaker containing Tyrodes solution at 37 Co.
Segments of ileum (2 cm in length) were
mounted in the 25 ml tissue bath and under 1 g
tension.
One end of the segment was attached to a
glass tissue hook and the other end was attached
by cotton thread to a force transducer coupled to
a transbridge and PowerLab Data Acquisition
System (Australia, ADInstruments) connected to
a computer running Labchart software (Version
7) for measurement of isometric tension.
A pre-load of 1 gm was applied to each tissue
and kept constant through the experiments. The
tissue was washed several times within 5 min.
interval and allowed to equilibrate for 30 min.,
before recording the isometric contractions by
submaximal concentration ACh induction. An
agonist constant time 20 seconds was used
together within 3 minutes interval between doses
once the tissue was stabilized with reproducible
effects from the doses of standard. The volumes
used were usually not exceeding 5% of bath
volume. Test materials were then added in a
cumulative pattern was to obtain concentrationdependent inhibitory responses (Van Rossum,
1963). The relaxation of ileum preparation, precontracted with high ACh 10 uM was expressed
as percent of the control response mediated by
ACh.
Isolated
Trachea
Experimental Protocol
Preparation
and
The trachea was removed, cleaned and 5
rings (each containing 3-4 cartilaginous ring
segments) were obtained from distal region of
the trachea. The tissue was suspended on two
stainless steel wires in 10 ml organ bath
containing Krebs solution at 37 Co under
isometric tension of 1 gm. The preparation was
allowed to equilibrate for at least 1 hr before the
addition of any drug, while, it was washed with
buffer solution every 15 min, the pH of the
buffer was maintained at 7.4 by continuous
aeration of the organ bath with a gas mixture
contain 95% O2 and 5% CO2.
In some preparations, ACh (10 uM) was used
to stabilize the respective preparations until
constant responses of each agonist were
produced.
After
obtaining
sustained
contractions, the relaxant effect of the test
materials were assessed by adding in a
cumulative fashion. Cumulative dose-response
curves for ACh were constructed using different
concentrations of agonist. When a 3-fold
increase in concentration produced no further
increment in response, the tissue was washed to
re-establish the base-line tension. Isometric
responses were recorded using force transducer
coupled to the transbridge and Power Lab Data
Acquisition System and computer running
Labchart software for measuring isometric
tension. Inhibition of contraction of the tracheal
ring was expressed as a percentage of the
maximum contraction.
3.602) and -3.620±0.0252 (-3.672 to -3.569),
whereas E-Max% values were 101.8 % and
99.09 %, respectively.
Fig 1. Cumulative dose-response curve for the effects of
Luteolin on KCl (60mM) induced contractions in rats aorta.
*** represents P<0.0001.
Statistical analysis
Data are presented as Means ± SEM. The
statistical analysis was performed using one way
analysis of variance (ANOVA) followed by
Dunnett test. P-value less than 0.05 (P<0.05)
was considered as statistically significant. The
Log IC50s values (i.e., the concentration of the
agonist or extract that produced 50% reduction
of maximal relaxant responses) were determined
from the concentration–response curves by nonlinear regression analysis using GraphPad
Prism™ software, version 6 (GraphPad
Software, Inc., San Diego, CA, USA).
RESULTS
Dose–response curves of luteolin in PE- and
KCl-Contracted Thoracic Aortic rings
Dose–response curve of the effect of
luteolin
against
KCl
and
PE-induced
contractions in aortic rings are shown in Fig (1
and 2). Luteolin at a concentrations from 2X10-4
– 4X10-4 M caused a highly significant
(P<0.001) relaxant effect on KCl precontracted
rat thoracic aortic rings. Luteolin also produced
significant relaxed effect on PE 10 uM induced
contraction in aortic rings 2X10-4 (P<0.05) and
3X10-4 – 4X10-4 M (P<0.001).
The Log IC50±SE, (Log IC50 of CI 95%)
and E-Max % of maximum contraction (E-Max)
are shown in Table 1. Luteolin showed more
potent inhibitory effect on KCl than PE-induced
contractions in aorta. For KCl and PE
precontracted aorta, Log IC50’s± SE (Log IC50
of CI 95%) were -3.635± 0.01655 (-3.669 to -
Luteolin on PE (10 uM) induced contractions in rats
aorta.*, ** and *** represent P< 0.05, P< 0.01 and
P<0.001, respectively.
Dose–response curves of Chlorogenic Acid in
PE- and KCl-Contracted Thoracic Aortic rings
chlorogenic acid against KCl and PE-induced
contractions are shown in Fig (3 and 4).
Chlorogenic acid at concentrations 4.5X10-4 –
5X10-4 caused a significant (P<0.01) relaxant
effect on KCl 60 mM precontracted rat’s
thoracic aorta. Chlorogenic acid also produced
significant relaxant effect on PE 10uM induced
contraction in aortic rings 3X10-4 (P<0.05) and
3.5X10-4 – 5X10-4 M (P<0.001).
222
aortic rings. The Log IC50± SE, (Log IC50 of CI
95%) and E-Max % are shown in Table 1.
Chlorogenic acid on KCl (60mM) induced contractions in
rats aortic ring. * and ** represent P<0.05 and P< 0.001,
respectively.
Amygdalin on PE (10 uM) induced contractions in rats
aortic rings.
The Log IC50± SE, (Log IC50 of CI 95%) and
E-Max % are shown in Table 1. Chlorogenic
acid showed inhibitory effect on KCl and PEinduced contractions in aorta. For KCl and PE
precontracted aorta, Log IC50’s± SE (Log IC50
of CI 95%) were -3.314±0.03266 (-3.380 to 3.247) and -3.466±0.007871 (-3.482 to -3.450),
whereas E-Max% values were 97.48 % and
106.4%, respectively.
Tannic acid on PE (10 uM) induced contractions in rats
aorta.
Dose–response curve of Amygdalin and Tannic
Acid in ACh-Contracted Rat Ileum
Chlorogenic acid on PE (10 uM) induced contractions in
rats aortic rings. *** represents P<0.0001
Dose–response curves of Amygdalin and
Tannic Acid in PE-Contracted Thoracic Aortic
rings
Amygdalin and Tannic acid against PE-induced
contractions are shown in Fig (5 and 6).
Amygdalin and Tannic Acid produced nonsignificant effect on PE-induced contraction in
223
Amygdalin and Tannic acid against on AChinduced ileal contractions are shown in Figures
(7 and 8). Amygdalin at (3 X 10-2 M), (1.8X10-21X10-2 M) and (6.7X10-3- 4X10-3 M) caused a
significant relaxation in the ACh (10 uM)
precontracted ileum of the rat at level (P<0.001),
(P<0.01) and (P<0.05) respectively.
Tannic acid at concentrations 3X10-4-7
3X10 M caused a highly significant relaxation
(P<0.001), whereas at 1X10-7 M, it caused just a
significant (P<0.05) relaxation in the ACh (10
uM) precontracted trachea of the rat.
The Log IC50± SE, (Log IC50 of CI 95%) and
E-Max % are shown in Table 1. The Amygdalin
and Tannic acid produced inhibitory effect on
ACh- induced contractions, with a (Log IC50±
SE) of -5.547±0.1742 M/mL and -5.421±0.2429,
(with a Log IC50 of CI 95% between -5.923 to 5.171 and -5.926 to -4.916) and E-Max of
63.89% and 68.87% respectively.
Fig. 9 . Cumulative dose-response curves for the
effect of Amygdalin on ACh (10 uM) induced
contractions in rats tracheal ring.
Fig 7. A cumulative dose-response curve for the effect of
Amygdalin on ACh (10 uM) induced contraction in rats
ileum. *** represents P<0.0001, ** represents P<0.001 and
*represents P<.0.05.
Fig. 10. Cumulative dose-response curves for the
effect of Tannic acid on ACh (10 uM) induced
contractions in tracheal rings
Fig 8. A cumulative dose-response curve for the effect of
Tannic acid on ACh (10 uM) induced contraction in rats
ileum. *** represents P<0.0001 and *represents P<.0.05.
Dose–response curve of Amygdalin and Tannic
Acid in ACh-Contracted Rat trachea
Amygdalin and Tannic acid against PE-induced
tracheal contractions are shown in Fig (9 and
10). Both amygdalin and tannic acid produced
non-significant effect on ACh (10 uM)
precontracted trachea of the rat. The Log IC50±
SE, (Log IC50 of CI 95%) and E-Max % for
amygdalin and tannic acid are shown in Table 1.
Discussion
The present study indicated that luteolin and
chlorogenic acid have potent relaxant effect on
KCl and PE-induced contractions on rat’s aortic
ring. PE is a selective alpha 1- adrenergic
receptor agonist and induced an initial transient
phasic contraction followed by a tonic
contraction (Shi et al. 2006). The initial
contraction is mediated by intracellular Ca++
release, whilst the sustained tonic contractions
resulted from Ca++ influx via the receptoroperated Ca++ channels through activation of Gprotein and activates Phospholipase C (PLC)
which elevate Ininisitol triphosphate and Ca++
(Karaki & Weiss 1988).
It is well known that KCl induces smooth
muscle contraction through activation of voltage
gated calcium channels (VGCs) and subsequent
release of Ca++ from the sarcoplasmic reticulum,
without influencing other signal transduction
systems such as phosphatidylinositol turnover
and Ca++ sensitization (Kumar et al. 2008).
224
The relaxant effect of Luteolin on isolated
tissue preparations used during the present work
may be partially due to the inhibition of protein
kinase C. Inhibition of cyclic nucleotide
phosphodiesterases
(PDE)
or
decreased
++
Ca uptake may also contribute to their
vasodilatory (Duarte et al. 1993).
The mechanisms of relaxant action of luteolin
in the current study are similar to those of (Ko et
al. 2005) and his coworker. They stated that
luteolin had antispasmolytic activity in isolated
guinea pig trachea, which may be due to its
inhibitory effects on both PDE activities and the
subsequent reduction in [Ca++]i of the
trachealis(Ko et al. 2005).
In thoracic aorta, the chlorogenic acid also
produced dose-dependent inhibiting effect on
contractions induced by PE and high K+ in
isolated thoracic aorta. It is difficult to compare
the results since the current study represents a
first attempt to study the effect of chlorogenic
acid on KCl and PE precontracted aorta rats.
However, (Bankar et al. 2011) found the
vasorelaxant and antihypertensive effects of
Cocos nucifera Linn, (rich in chlorogenic acid)
via nitric oxide production in a concentration
and endothelium-dependent manner This effect
may due to direct activation of nitric oxide /
guanylate cyclase pathway, stimulation of
muscarinic receptors and/or via cyclo-oxygenase
pathway.
Amygdalin and tannic acid showed a nonsignificant effect on aorta and trachea while have
a significant effect on ileum rats. Since no
attempt have been made so far to study the effect
of amygdalin and tannic on rat ileum, trachea
and thoracic aorta pretreatment with ACh and
PE it is in almost impossible to compare the
results.
Amygdalin and tannic acid inhibited the ACh
(10 µM) induced contraction in a concentration
dependent fashion in rat’s ileum. ACh is a
neurotransmitter that activates muscarinic
receptor located in the plasma membrane of
smooth muscle cells and bind with M3 receptor
(Chung 2008). After binding of ACh with its
receptor, it activates G-protein (Gq/p) and
activates PLC.
The PLC cleaves PIP2 into diacyle glycerol
(DAG) and IP3. IP3 IP3 releases Ca2+ from the
endoplasmic reticulum (ER), and DAG activates
PKC (Chung 2008). ACh caused depolarization
and tonic contraction of intestinal and tracheal
smooth muscle (Chung 2008).
225
Activation of muscarinic receptors of
longitudinal smooth muscle of rat’s ileum and
trachea increased the frequency of action
potential and rising rate of depolarization which
results in smooth muscle contraction (Reddy et
al. 1995). The ACh-evoked contraction is
generally considered as mediated via M3
muscarinic
receptor.
Furthermore,
the
preponderance of M2 subtype muscarinic
binding sites may contribute in ACh induces
contraction (Fatehi et al. 2004).
In ileum, the amygdalin and tannic acid may
act through blocking the muscarinic receptors.
The anti-contractility effect of the amygdalin
and tannic acid on rat ileum may be due to
inhibit either non-selective cation channels in the
plasma membrane, which results in membrane
depolarization or inhibits activation of the
release of intracellular Ca++. Change in the
membrane potential stimulates Ca++ influx
through VGCs (Namkung et al. 2010). However,
(Calixto et al. 1986) showed that tannic acid can
affect Ca+ availability for contraction of smooth
and cardiac muscles. This action could well
mask the effects of other active constituents of
tannic-rich plant extracts (Habauzit & Morand
2012).
In conclusion, this summary of existing
scientific evidence indicates that inhibitory
effect of luteolin and chlorogenic acid on the
contraction induced by PE and KCl may be due
to their anti-adrenergic and anti-hyperpolarizing
effect.
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226
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227
IMPACT OF NUTS CONSUMPTION ON BLOOD PRESSURE, GLYCEMIC
STATUS, LIPID PROFILE AND TOTAL PROTEIN IN HEALTHY HUMAN
VOLUNTEERS
*
Suad Y. AL-Kass*, Omar A. M. Al-Habib and Kajeen H. Jasim
Dept. Biology, Faculty of Science, University of Zakho, Kurdistan Region-Iraq
Dept. Biochemistry, School of Basic Sciences, Faculty of Medical Sciences, University of Duhok, Kurdistan Region-Iraq.
(Accepted for publication: July 9, 2013)
Abstract
Objective:
To investigate the advantage of consumption of a mixture of almond and pistachio on blood pressure,
lipid profile, sugar and protein levels in healthy volunteers.
Subjects and Methods:
This dietary intervention study was carried out during the period from February to March 2013. A
total of 48 apparently healthy males students from Military Academy / Zakho, Kurdistan Region of Iraq
participated in this study. All the students were living in a controlled environment. Blood samples were
taken and analyzed for the determination of serum glucose, lipid profile, total protein, albumin and
globulin. Parameters were obtained at baseline, 3 and 6 weeks after daily consumption of 50gm of almond
and pistachio mixture.
Results:
The results of the current study demonstrated that daily consumption of 50 gs of almond and
pistachio mixture for 3-6 weeks significantly ( P<0.05 – 0.001) decreased the diastolic blood pressure and
the level of blood glucose , whereas body mass index (BMI) was not influenced at all. Serum total protein,
albumin and globulin levels were significantly increased (P<0.05-0.005).
Finally, Total cholesterol (TC), triglycerides (TG), low density lipoprotein-cholesterol (LDL-ch), very
low density lipoprotein cholesterol ( VLDL-ch), TC/HDL and LDL/HDL were significantly
decreased (P<0.05-0.005) after 6 weeks of nuts consumption, where as high density lipoproteincholesterol, was significantly increased (P<0.01).
Conclusion:
This dietary intervention trial, demonstrated that almonds and pistachio mixture improved blood
glucose , total protein, and lipid profile to much better levels than that obtained previously using each
one separately in healthy volunteers.
Introduction:
Epidemiologic studies showed that frequent
nut consumption decreases the risk of coronary
heart disease (CHD) (López-Uriarte et al, 2010).
Compared with people who consumed nuts less
than one time per week, people who eat nuts at
least five times per week showed 50% reduction
in the risk of CHD. Furtherer more, it also
represents a good source of dietary fiber,
vitamins, micronutrients, antioxidants, and the
amino acids arginine (Sari et al, 2010). Studies
have shown that a diet low in saturated fatty
acids (SFAs) and cholesterol tends to decrease
the risk of heart disease (Vadivel et al, 2012).
Foods, such as nuts, are rich in monounsaturated
(MUFAs) or polyunsaturated fatty acid (PUFAs)
have been recommended as substitutes for high
SFAs food to reduce risk of coronary heart
disease by favorably altering cholesterol levels
in the body (Srinath, 2003). Nuts have been
shown to decrease biomarkers of oxidative stress
(such as oxidized LDL cholesterol) and improve
the blood lipid profile (Vadivel et al, 2012) and
have a significant role in decreasing
inflammation processes, improving endothelial
function, and insulin sensitivity (Salas-Salvadó
et al, 2011). In addition to antioxidant-rich
foods, HDL-ch has been associated with
improvement of lipid oxidation, as well as antiinflammatory and antithrombotic activities
(Salas-Salvado´ et al, 2011). Elevated HDL-C
level is associated with reduced oxidative stress
(Chiavaroli, 2010).
Consumption of nuts has not been linked to
weight gain despite increases in caloric intake
caused by its consumption (McBride, 2011).
Since an inverse relationship was observed
between the frequency of nut consumption and
228
body mass index (BMI) (Fitschen, 2010), and its
consumption, have shown to help in maintaining
body weight (Tsantili et al, 2011). It was found
that nuts reduce the glycaemic impact of
ingested carbohydrate rich foods, due to their
high fibre and unsaturated fat content (SalasSalvadó et al, 2008). Nuts are potentially
protective foods against high blood pressure
(BP) because they are low in sodium, and
contain significant amounts of mono- and
polyunsaturated fatty acids, minerals (such as
magnesium, potassium and calcium), dietary
fiber, and antioxidants, and all these components
might interact to improve blood pressure
(Kleopatra and Katsilambros, 2011). Nuts such
as almonds and pistachios are rich in several
beneficial compound, such as omega-3- fatty
acids. Also it has been proposed that the
bioactive compounds in nuts may help in
lowering the risk factors of cardiovascular disese
by improving endothelial function and BP, in
addition to lowering oxidative stress and
inflammation (Soliman, 2012).
In this study we therefore investigated
the effect of a mixture of almond and pistachio
on lipid parameters, total protein, glucose, blood
pressure and oxidative status in healthy
volunteers living in a controlled environment.”
Subjects and Methods :
Subjects:
Forty-eight male volunteers, mean age 22
years between 18 and 36y old, they are student
from Military Academy / Zakho, Kurdistan
Region of Iraq under controlled environment,
having the same diet and living in the same
place.
Also they have a regular wake/sleep hour
and same daily activity. They were not provided
by any additional food other than the dietary
recommendations given to the volunteer, with
free access to water.
The enrolled subjects were healthy and free
of acute or chronic medical disorders with no
family history (father and / or mother) of heart
diseases. Their bodies were also
normal.
Furthermore, all subjects underwent a
detailed
physical
examination
by
the
physician and the detailed medical history for
each was taken. Exclusion criteria were
smoking, consumption of alcohol, history of
eating nuts frequently (more than once a week),
a history of food or nut allergy, and regular use
of any medications including vitamin
229
supplements. The volunteers were informed
about the nature of the study and a written
consent was then obtained from each subject.
Study Design:
The study design included hree controlledfeeding periods. The first group, a run-in period
preceded the test diet to establish a baseline
for regular meals prepared in the Academy
kitchen. These included all major food groups
but did not include nuts. The amount of food
was standardized for each volunteer. The second
and third groups, included the addition of 50 gm
of a mixture of almond and pistachios (25gm of
each) consumed with controlled diet for three
and six weeks, respectively. The students were
instructed to eat their daily ratio of nuts in the
morning with or after breakfast. During the six
week of the study, the students were allowed to
go outside the academy for only a single day.
Since the academy programme
and
the
activities of the volunteers have been preplanned for the entire year of education, threre
was no change in their daily activities during the
study period. Parameters were obtained at
baseline, three and six weeks after daily
consumption of 50gm of a mixture of nuts.
Data Collection:
A pre-tested questionnaire was designed to
obtain information on age, anthropometric
measurements, smoking, alcohol and nut
consumption, type of diet (vegetarian or mixed),
family history of diseases, past medical history
and any medication if available. Blood pressure
was measured, fasting serum glucose, lipid
profile, total protein, albumin and globulin were
assayed and body mass index was calculated.
Collection of blood samples:
Venous blood samples (10 ml) were collected
between 7.00- 9.00 a.m after12-14 hour fasting
by using sterile disposable syringe. Care was
taken to avoid venous stasis during sample
withdrawing and the blood was added into a
clean capped disposable tube and transferred in
cooling ice bag to Zakho Hospital Laboratories
for biochemical analysis. The blood samples
were then centrifuged (HITASHI model O5P21) at 3000 rpm for 10 minutes to separate
serum from the clot. The obtained serum was
divided into 5 parts in eppendorf capped tubes
frozen at -28 oC until the time of analysis.
Biochemical Analysis:
Results:
Total protein and serum albumin levelsd were
measured colorimetrically using standard kit
(Biolabo reagent, France). Globulin level was
estimated mathematically by subtracting
albumin from total protein. Total cholesterol,
serum TG and serum HDL-ch were determined
by an enzymatic colorimetric method using
commercial kits (Biolabo, Maizy, France). Very
low density lipoprotein cholesterol VLDL
cholesterol concentration was calculated and
LDL-C was calculated mathematically from the
total cholesterol, the TG and HDL-ch
concentration by the Friedewald,s formula.
Serum glucose was determined by an
enzymatic colorimetric method using a
commercial kit supplied by Biolabo (Maizy,
France).
The results of the effect of consumption
of a mixture of almond and pistachio on BMI,
blood pressure, glucose and proteins levels are
shown in (Table 1 and Figures1,2,3). As the
results indicate, the BMI values were slightly,
but nonsignificantly (P > 0.05) reduced after
nuts consumption for three and six weeks.
Nuts consumption influenced the systolic and
diastolic blood pressure to a different extent.
Thus, nuts consumption for 3 and 6 weeks nonsignificantly ( P > 0.05 ) reduced the systolic
blood pressure, whereas
the diastolic blood
pressure was significantly (P<0.001) reduced
after 3 weeks of nuts consumption which
remains more or less stable after further
extension of nuts consumption to 6 weeks.
Nuts consumption for 3 weeks nonsignificantly ( P > 0.05) reduced the level of
serum glucose, while extension of the period of
nuts consumption to 6 weeks significantly ( P <
0.001 ) reduced the level of serum glucose (
table 1 ). Nuts consumption for 3 weeks
significantly ( P < 0.001) increased the level of
serum albumin, where as nuts consumption
for 6 weeks produced significant (P< 0.05)
elevation in the levels of serum total serum
protein and globulin ( Table 1 ).
Statistical Analysis:
All data were analyzed using the statistical
package for social sciences SPSS version 20
software for windows 7. The results were
expressed as mean ± standard deviations (mean
± SD). One way ANOVA-test was used to
compare parameters in different studied groups.
P-values (P ≤ 0.05) were considered to be
statistically significant.
230
Table.1: The effect of consumption of a mixture of almond and pistachio at baseline , three weeks and six
weeks on Body Mass index, Blood Pressure, serum Glucose and Proteins.
Variables
BMI
2
(kg/m )
Baseline
3
6
N=48
Mean ±SD
Group 1
weeks
N=4
8
Mean ±SD
weeks
N=4
8
Mean ±SD
P –value
25.34±3.00
24.29±2.57
24.23±2.22
Systolic BP
(mm Hg)
120.8±6.27
119.8±6.40
118.8±4.68
Diastolic BP
(mm Hg)
79.8±3.99
76.7±4.63
76.3±2.16
0.001*
Serum glucose
(mg/dl)
91.28±7.29
88.33±7.17
85.90±6.36
0.001*
Serum total protein
(g/dl)
7.16±0.41
7.27±0.33
7.33±0.31
0.050*
Serum Albumin
(g/dl)
4.61±0.10
4.67±0.11
4.69±0.08
0.001*
Serum Globulin
(g/dl)
2.55±0.31
2.6±0.22
2.64±0.23
0.02*
0.067
0.2
4
*Means the presence of a significant difference (one way ANOVA).
Figure .1: The relationship between the period of nuts consumption and the percent of change in Blood
pressure.
*= significant according to one way ANOVAs test.
NS= nonsignificant according to one way ANOVAs test.
231
Figure .2: The relationship between the period of nuts consumption
and the percent of change glucose and BMI
*= significant according to one way ANOVAs test.
NS= nonsignificant according to one way ANOVAs test.
Figure.3: The relationship between the period of nuts consumption and the percent of change in
Protein parameters
232
Serum Lipids:
The effect of nuts consumption baseline, three and six weeks on serum lipid profile are shown in
Table 2 and Figure 2. The effect of nuts consumption on lipid profile parameters was not in the same
direction. Thus, the levels of triglycerides, total cholesterol, LDL-ch, VLDL, the ratios of TC/HDL
and LDL/HDL were significantly (P< 0.05 - 0.01) reduced after 6 weeks of nuts consumption, where
as the level of HDL- ch was significantly ( P < 0.05) increased from 50.87±6.82 to 54.75±6.06 after
six weeks of nuts consumption. However, the levels of change in the above parameters after three
weeks of nuts consumption were statistically not significant.
Table 2: The effect of consumption of a mixture of almond and pistachio at Baseline , 3 and 6 weeks on lipid
profile parameters (Mean ± SD) in human subjects.
Baseli
Variables
ne
N=
48
Mean ±SD
Group 1
3
6
week
N=
48
Mean
±SD
Group 2
week
N=4
8
Mean ±SD
Group 3
P –value
Total cholesterol
(mg/dl)
180.71±18.95
174.74±19.27
169.03±18.37
0.012*
Triglyceride
(TG) (mg/dl)
103.96±17.71
100.83±17.13
95.29±15.09
0.039*
HDL-ch
(mg/dl)
50.87±6.82
52.74±6.21
54.75±6.06
0.013*
LDL-ch
(mg/dl)
109.04±10.95
101.82±10.7
95.18±8.25
0.015*
VLDL-ch
(mg/dl)
20.79±5.34
20.18±5.11
19.10±4.18
0.008*
T.C/HDL
(mg/dl)
3.75 ± 2.77
3.51±3.10
3.08±3.03
0.002*
LDL/HDL
(mg/dl)
2.34 ± 1.60
2.03±1.72
1.73±1.36
0.004*
233
Figure 4: The relationship between the period of nuts consumption and the percent of change in lipid profile
parameters.
* Means the presence of a significant difference (one way ANOVA ).
DISCUSSION
Recent nutritional research had focused on
the consumption of nuts, including almond and
pistachio, as a part of heart healthy diet due to its
protective effect by reducing the risk of heart
disease and coronary risk factor (Griel and KrisEtherton, 2006). Most nuts are rich in
monounsaturated
fatty
acids,
essential
polyunsaturated fatty acids, linoleic acid and α –
linolenic acid. Furthermore, they also contain
bioactive components such as plant proteins,
fibers, tocopherols, phytosterols, phenolic
compounds, arginine and other bioactive
compounds that collectively contribute to their
cardioprotective, antiobesity, antioxidant and
anticancer effects (Kris- Etherton, et al, 2008).
The results of the current study showed that
consumption of a mixture of almond and
pistachio for 6 weeks did not produced any body
weight gain with no changes in BMI. These
results are consistent with those reported by
(Jaceldo-Siegl et al, 2011; Vadivel et al, 2012;
Wang et al, 2012).
Martinez-Gonzalez and Bes-Rastrollo, (2011)
reported that high or recommended doses of
daily consumption of pistachio for 12 weeks
resulted no changes in BMI. Furthermore, it
has been indicated that nuts consumption
produced beneficial effects against body weight
gain and adiposity (Mattes et al, 2008). Several
physiological mechanisms may explain the lack
of body weight gain observed in association with
nut consumption, since it induce satiation
(reduction in total amount of food eaten in a
single meal ) and satiety ( reduction of the
frequency of meals) (Martinez- Gonzalez and
Bes- Rostroll, 2011). High energy provided
with nuts can be compensated by a lower energy
intake in meals (Cyril et al,2010). In addition,
the presence of certain bioactive compounds in
nuts, including polyphenols, is assumed to have
beneficial roles in body weight control (Richard
et al, 2008).
The results of the current study showed that
consumption of a mixture of almond and
pistachio for six weeks had favorable effects on
blood pressure, especially diastolic blood
pressure which was significantly reduced. These
results are in agreement with those of CasasAgustench et al, (2011a and b). The later
indicated that due to pistachio contain high level
of potassium, clinical studies suggested that
increased potassium intake may help in
controlling blood pressure in normal and
hypertensive subjects (Baer et al, 2012).
Reduction in diastolic blood pressure indicates
that the active ingredients of nuts caused
significant vasodilatation which ultimately
lowers the diastolic blood pressure. This
vasodilatation effects may be partly attributed to
the presence of high potassium which usually
cause vasorelaxation (Al-Surchi, 2010).
Nuts consumption
reduces the overall
glycemic index of the diet, when added to meals
234
rich in carbohydrate. Nuts also slow down the
passage of the meal through the gut and reduce
blood glucose levels following the meal (Cyril et
al,2010). The results of the present study showed
that consumption of a mixture of almonds and
pistachio for six weeks significantly decreased
the level of blood glucose. This finding is
compatible with other studies (Cohen and
Johnston, 2011).
Wang et al,(2012) indicated that, when
pistachio nuts taken together with high
carbohydrate foods, it decreased the absorption
of carbohydrate and lowered postprandial blood
glucose. Furthermore, the addition of almonds
or pistachios to a meal can reduce blood glucose
and insulin levels following the meal (Kendall et
al,2011).
The results of the present study showed
significant increase in total protein, albumin and
globulin after consumption a mixture of almond
and pistachio with meals for six weeks.
Comparison of the studied groups with each
other, showed that albumin level was
significantly increased during the 1st period by
1.3%, globulin fraction increased when group2
and 3 compared to group 1 by 1.3% and 1.7%,
respectively. Total protein was also increased
when group 3 was compared with group 1 by
2.3%. These data are considered as new one
since it represent the first attempt to study the
effect of a mixture of almond and pistachio on
total protein, albumin and globulin. Thus these
results cannot be compared since no previous
studies have been carried out in any of the
intervention trial on the effect of nuts
consumption especially those studies which
related to almond or pistachio.
In the present study, consumption of a
mixture of almond and pistachio for six weeks
significantly reduced the levels of TG, TC, LDLcholesterol, VLDL- cholesterol, TC/HDL and
LDL/HDL, where as, the level of HDLCholesterol was significantly elevated. These
results are in agreement with those of Karen et
al, (1999) who found a significant decrease in
total
cholesterol,
total
cholesterol/HDL
cholesterol, LDL/HDL cholesterol and an
increase in HDL cholesterol after pistachio
nuts ingestion. The favorable fatty acid
composition and lipid lowering effect of nuts
have been demonstrated in experimental studies
with almonds and pistachios (Yang et al,2009).
The effect of the nuts mixture on lipid profile
parameters take longer time than that is needed
for antioxidants and pro-oxidants to exert their
235
effects (Shih et al,2010). The results of the
present study are unique with respect to the
improvement of TG level
which was
significantly decreased by following nuts
consumption. Furthermore, it also showed that
the percent change for any parameters were
more than that obtained from other studies (Sari
et al,2010). Finally, the ratios of TC/HDL and
LDL/HD were decreased by 17.8% and 26%,
respectively. This percent of change is much
higher than that obtained by others researchers
(McBride,2011), which can be considered as
better predictors of coronary heart disease than
changes in LDL alone. Accordingly, it can
be concluded that the selected mixture of nuts is
good and can be considered as a favorable heart
healthy mixture.
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238
STUDIES ON SOME ENZYME ACTIVITIES IN LAMINATED AND
GERMINAL LAYERS OF HYDATID CYSTS ISOLATED FROM DIFFERENT
INTERMEDIATE HOSTS IN ZAKHO, DUHOK PROVINCE, KURDISTAN
REGION OF IRAQ.
Wijdan M.S. Mero and Araz R.I. AL Bosely
Department of Biology, Faculty of Science, University of Zakho, Kurdistan Region - Iraq
Abstract:
The current study deals with some enzyme activities in laminated and germinal layers of hydatid cysts
isolated from liver and lungs of infected sheep, goats and cattle slaughtered in Zakho abattoirs and cysts
isolated from humans. The activities of the enzymes, acid phosphatases (ACP), alkaline phosphatase
(ALP), lactate dehydrogenase (LDH), glutamate oxaloacetate transminase (GOT) and glutamate Pyruvate
transaminase (GPT) were measured in cysts isolated from both liver and lungs of infected sheep, goats,
cattle and humans. The activities of all of these enzymes were higher in laminated layer as compared with
their activities in germinal layer, however in general infected host tissue showed the highest enzymatic
activities as compared with hydatid cyst.
c
I. INTRODUCTION
ystic Echinococcosis (CE) or hydatid
cyst (HC) is a zoonotic disease of
cosmopolitan distribution caused by the larval
stage of the Echinococcus granulosus (Schantz,
1991, Eckert et al., 2000 and Eckert & Deplazes,
2004). Human can be infected with the larval
stage when ingests the eggs of the parasite either
with food or drinks (Thompson, 1986). There are
six species belonging to the genus Echinococcus.
Four of them are infective to human causing
various forms of Echinococcosis (WHO, 2001).
These are E. granulosus, causing cystic
Echinococcosis (CE); E. multilocularis, causing
alveolar Echinococcosis (AE); E. vogeli and E.
oligarthrus
both
causing
polycystic
Echinococcosis (D’Alessandro, 1997).
E.
granulosus is the most common of the four
species. E. multilocularis is rare but is the most
virulent; and E. vogeli is the most rare
(Moldovan et al., 2012). Despite to the
economical and medical importance of
hydatidosis, little attention has been paid to the
comparative study of the parasite and its host
metabolism. The metabolic pathways may vary
in different parasitic species and in their hosts,
MacPherson et al. (1985) proposed a strategy for
the chemotherapy of infectious diseases utilizing
biochemical differences, and stated that the
inhibition of enzyme systems that are crucial to
the parasites but not the host may be the basis of
rational approach to the chemotherapy of the
parasite. However, biochemical studies are also
useful in differentiating strain variations of E.
granulosus in different countries (Radfar and
239
Iranyar, 2004). The strain characterization is
particularly important in regions where more
than one species of livestock intermediate host
exists and where there is the possibility of
different cycles of transmission and sources of
infection for humans (Thompson and Lymbery,
1995). The present study was designed to
evaluate the activities of some enzymes in
laminated and germinal layers of the cyst and the
host tissues which are in direct contact with the
cyst and those which are at a distance of 5 cm
from the cyst wall.
II. MATERIALS AND METHODS
During this study, a total of 40 HCs along
with the tissues of infected organs were collected
from infected animals (14 sheep, 14 goat and 12
cattle). In addition, 14 humans HCs were
obtained after surgical removal of cysts from
liver and lungs of patients at Azadi Teaching
Hospital in Duhok city and Zahko hospital. Also
12 samples from organs (liver and lung) of
uninfected sheep, goats and cattle were collected
to be used as control. The activities of the
following enzymes were studied acid
phosphatases (ACP), alkaline phosphatase
(ALP), lactate dehydrogenase (LDH), glutamate
oxaloacetate transminase (GOT) and glutamate
Pyruvate transminase (GPT) using enzyme kits
supplied by Biolabo reagents (France). The
Buffer systems (Citrate buffer, CarbonateBicarbonate buffer and Tris-HCl buffer). For
tissue homogenization 0.5 gram of germinal and
laminated layers, infected tissues in direct
contact with cyst wall, tissues at 5 cm distance
of the cysts and uninfected tissue were used.
The sample were cut in to small pieces
homogenized with 5 ml of buffer solution using
glass homogenizer connected to a variable speed
stirrer and placed in a beaker containing crushed
ice (Mero et al., 1988). After complete
homogenization, the extract was centrifuged at
4000 rpm for 15 minutes and the supernatants
were kept in labelled sample tubes and stored in
a deep freezer at -40°C until used. The activities
of the enzymes were determined using
spectrophotometric method (Jenway 6300,
England).
III. RESULTS AND DISCUSSION
1. The result of the activity of Acid phosphatase
(ACP) in hydatid cyst isolated from sheep, goats,
cattle and human liver and lungs are shown in
Table (1). It is obvious that the activity of Acid
phosphatase (ACP) in laminated layer was
higher than in germinal layer in all cysts,
isolated from both liver and lungs of the studied
hosts with the highest being in infected tissue as
compared with hydatid cyst and uninfected
tissue.
Table (1): The activity of ACP enzyme in HC layer and host tissues.
2. The result of the activity of glutamate pyruvate transminase (GPT) in hydatid cyst isolated from
sheep, goats, cattle and human liver and lungs are shown in Table (2). The activity of GPT in
laminated layer was higher than that of germinal layer in all cysts isolated from both liver and lungs of
all intermediate hosts with the highest being in infected tissue as compared with cyst and uninfected
tissue.
Table (2): The activity of GPT enzyme in HC layer and host tissues.
240
3. The result of the activity of glutamate oxaloacetate transminase (GOT) in hydatid cyst isolated from
sheep, goats cattle and human liver and lungs are shown in Table (3). The activities of GOT in
laminated layer was higher than that of germinal layer in all cysts isolated from both liver and lungs of
all intermediate hosts with the highest being in infected tissue as compared with cyst and uninfected
tissue.
Table (3): The activity of GOT enzyme in HC layer and host tissues.
4. The result of the activity of alkaline phosphatase (ALP) in hydatid cyst isolated from sheep, goats,
cattle and human liver and lungs are shown in Table (4). The activity of ALP in laminated layer was
higher than that of germinal layer in all cysts isolated from both liver and lungs of all intermediate
hosts with highest being in infected tissue as compared with cyst and uninfected tissue.
Table (4): The activity of ALP enzyme in HC layer and host tissues.
241
5. The result of the activity of lactate dehydrogenase (LDH) in hydatid cyst isolated from sheep, goats,
cattle and human liver and lungs are shows in Table (5). It is obvious that the activity of LDH in
laminated layer was higher than in germinal layer in all cysts, isolated from both liver and lungs of the
studied hosts with the highest being in infected tissue as compared with hydatid cyst and uninfected
tissue.
Table (1): The activity of LDH enzyme in HC layer and host tissues.
The current study indicated that the activities
of all studied enzymes were high in laminated
layer in all studied hosts as compared to
germinal layer. Regarding host tissues, the
tissues in contact with hydatid cysts showed the
highest enzymatic activities. As hydatid cysts are
space occupying lesions, their growth exerts
pressure on the surrounding tissues which leads
to tissue damage and the leak of enzymes from
damaged cells to the surrounding tissue
(Zeheer,1997, Nyblom et al., 2004).
So far there is no any available studies
concerning the activities of these enzymes in
cyst layers, howevere, the available studies were
performed on protoscolices and hydatid fluid in
which high enzymatic activities were observed
(MacManus and Bryants (1995), Frayha and
Haddad (1980), Izadi & Ajami (2006), Rahdar
et al.( 2008) and Rouhani and Vatankhah (2008)
on HF and Abdullah (2010) on protoscolices).
References
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comparative enzymatic and total protein
content of hydatid cyst of Echinococcus
gernulosus isolated from Sheep and Goats
in Duhok province, Kurdistan Region of
Iraq. M.Sc. Thesis, College of Education,
University of Duhok, Iraq.
D’Alessandro,
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(1997),
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(2000), Echinococcosis: an emerging or
reemerging zoonosis? Int. J. Parasitol., 30:
1283-94.
Eckert, J. and Deplazes, P. (2004), Biological,
epidemiological, and clinical aspects of
echinococcosis, a zoonosis of increasing
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Comparative chemical composition of
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Echinococcus granulosus (Cestoda). Int. J.
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Izadi, J. and Ajami, A. (2006): Biochemical
profiles of hydatid cyst fluids of
Echinococcus granulosus of human and
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Camel). J. Animal Vet. Adv., 5(7): 574577.
MacPherson, C. N. L., French C. M., Stevenson,
L. K. and Arundel, J. H. (1985), Hydatid
disease in the Turkana District of Kenya,
IV. The prevalence of E. granulosus
infection in dog and observation on the
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McManus, D. P. and Bryant, C. (1995):
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of
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Echinococcus and Hydatid Disease,
Thompson R.C.A. & Lymbery A. J., eds.
CAB International, Wallingford, UK,
135–182.
Mero, W. M., Al-Zako, S. S. and Zakaria, S. J.
(1988), Comparative study on some
enzyme activity in Fasciola hepatica,
Fasciola gigantia and Bovine liver. J.
Edu. And Sci. Vol. 8. Dep. of Biology,
College of Science, University of MosulIraq, p 62-67.
Moldovan, R., Neghina, A. M., Calma, C. L.,
Marincu, I. and Neghina, R. (2012),
Human cystic Echinococcosis in two
south-western
and
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7-year
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Acta Trop., 121(1):26-9.
Nyblom, H., Berggren, U., Balldin, J. and
Olsson, R. (2004): "High AST/ALT ratio
may indicate advanced alcoholic liver
disease rather than heavy drinking".
Alcohol Alcohol, Pp.: 336-339.
Radfar, M. H. and Iranyar, N. (2004),
Biochemical profiles of hydatid cyst fluids
of Echinococcus granulosus of human and
animal origin in Iran. Vet. Arhiv., 74 (6):
435-442.
Rahdar M. H., Maraghil, S., Rafei, A. and
Razijalali, M. (2008): Comparison of
some electrolytes in hydatid cyst fluid and
serum of liver hydatidosis of sheep.
Jundishapur Journal of Microbiology,
1(1): 10-14.
Rouhani, S. and Vatankhah, A. (2008):
Biochemical Changes in the Fertile and
Sterile of Hydatid Cyst Fluid in Sheep.
Shahid Beheshty University of Medical
Sciences, Tehran, Iran, Parasitology
Department Pasteur Institute of Iran,
Tehran, 13th International Congress on
Infectious Diseases Abstracts, Poster
Presentations, p.23-65.
Schantz P. M. (1991),Parasiticzoonoses in
perspective. Int. J. Parasitol., 21: 161-70.
Thompson, R. C. A. (1986), Biology and
systematics of Echinococcus. In: R. C. A
THOMPSON. (ed): The biology of
Echinococcus and hydatid disease. George
Allen and Unwin, London, 5-43.
Thompson, R. C. A. and Lymbery, A. J. (1995),
Echinococcus and hydatid disease, 1st ed.
Wallingford, CAB International, 122-135.
WHO (2001), Office international des
epizooties, Eckert, J., Gemmell, M.A.,
Meslin, F. X., Pawlowski, Z.S.,
Echinococcosis in Humans and Animals: a
Public Health Problem of Global Concern.
Paris, France, 1–265.
Zeheer, A. (1997): Some biochemical changes in
Sheep hepatocttes surrounding natural
unilocular hydatd cysts. University of
Punjab. Ph.D. Thesis. Department of
Zoology, Pp.:131-134.
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244
HISTOPATHOLOGICAL STUDY OF THE EFFECT OF CISPLATIN ON THE
OVARIES OF FEMALES' ALBINO MICE
Intissar Numman Waheed*, Aveen Abdullah Mohammed Ameen**, Unce Jasib Jasim*** and Bakhtyar Nader Ali **
* Department of Biology, Faculty of Science, University of Zakho, Kurdistan Region-Iraq.
** Scientific Research Center, Faculty of Science, University of Duhok, Kurdistan Region- Iraq.
*** School of Medicine, Faculty of Medical Sciences, University of Duhok, Kurdistan Region-Iraq.
(Accepted for publication: July 10, 2014)
ABSTRACT:
Cisplatin is a chemotherapeutic agent used for treatment of solid tumors and is asserted as an
intermediately gonadotoxic agent. The present study was designed to study the cytotoxic effect of cisplatin
on the ovaries of females' albino mice. Twenty adult albino females' mice (11 weeks old, with 25gm
average weight) were randomly divided into two groups. Group (1) injected i.p. 0.025mg cisplatin\kg B.W.
as a single dose, group (2) control. Ten females (five from each group) were dissected after 6 days from
injection, while the remaining ten females were dissected after 12 days. Then the cytotoxic effect of
cisplatin on both ovarian follicles was evaluated by separately counting each follicle in the histological
sections, in addition to study their histopathological changes. The results showed that treatment with
cisplatin caused a significant decreased number of ovarian follicles compared with control group and this
effect was increased with time after administration. The histopathological study revealed that cisplatin
caused damaged in both cortical ovarian follicles and medulla layers. These damages included the
detachment of tunica albugina from the ovarian tissues; congestion of blood vessels, pyknosis, shrinkage
and deforming of the follicular ova; pyknosis of granulosa cells and their detachment from the basement
membrane; disorganization of granulosa cells in addition to disruption of their intracellular contacts with
oocytes and destruction of theca cells. In conclusion: treatment with single dose of cisplatin caused
damage and significant decrease in number of ovarian follicles which may result in females' infertility.
Keywords: Cisplatin, ovary, chemotherapy, mice, morphology.
Introduction
Chemotherapy is one of the major therapeutic
modalities commonly used for treatment of a
variety of cancers. In addition to the fact that
chemotherapy or radiotherapy alone cannot
achieve a satisfactory therapeutic outcome in
many cases, namely the complete remission of
tumors, these therapeutic agents may induce
severe side effects at therapeutically effective
doses (Park et al., 2009)
Cisplatin
(Cisplatinum
or
cisDiamminedichloroplatinum II) has become a
major chemotherapeutic agent in clinical
treatment of tumors since it was found to have
anti-cancer activity for the last two decades
(Hanign and Devarajan, 2003). It is used
particularly for treatment of solid tumors, such
as testicular cancer, ovarian cancer, bladder
cancer, cancer of the head and neck and smallcell lung carcinomas as well as solid tumors
resistant to other therapeutic regimens' (Loehrer
and Einhorn, 1984; Hanign and Devarajan,
2003). Cisplatin damages tumors via induction
of apoptosis, mediated by the activation of
various signal transduction pathways, including
calcium signaling, death receptor signaling, and
245
the activation of mitochondrial pathways (Florea
and Büsselberg, 2011).
Despite its clinical usefullness, cisplatin
treatment has been associated with several toxic
side
effects,
including
nephrotoxicity,
neurotoxicity and ototoxicity (Rabik and Dolan,
2007). Cisplatin is asserted as an intermediately
gonadotoxic agent (Tangir et al., 2003).
Moreover, it has been demonstrated that
cisplatin-associated infertility is caused by the
toxic effect on the primordial follicles. Since the
primordial follicles are not able to regenerate,
the damage caused by the exposure to toxic
agents may lead to ovarian insufficiency and
infertility (Yucebilgin et al., 2004). The severe
adverse effects occurring during cancer
chemotherapy restrict the appropriate use of
anticancer drugs (Jemal et al., 2003). The anticancer drugs, particularly those used in early
childhood and in reproductive period may cause
several complications such as ovarian
insufficiency and infertility (Sommezer and
Oktay, 2004). Therefore, in recent years, trials
have been initiated on several methods to
prevent
infertility
in
patients
given
chemotherapy (Oktay et al., 2005).
El-Sayyad et al., (2009) investigate the
effects of cisplatin, belonging to different
chemical classes on mice ovary toxicity. They
showed that drugs induced drastic abnormalities
with respect to body and ovary weight changes
as well as the histology of ovarian tissue. They
also identified pathological features at both
structural and ultrastructural levels, which could
be used to adjust the dose and duration of
treatment. Yeh et al., (2011) demonstrated that
the administration of cisplatin prior to pregnancy
caused significant reductions in fertility and fetal
outcomes. So for these reasons, the present study
was designed to study the toxic effects of
cisplatin on the ovaries of female albino mice.
Materials and methods
Animals and experimental design
Twenty adult female albino mice (Mus
musculus) of strain Balb/C (11 weeks old with
average weight 25gm) were obtained from
Animal Breeding House, Faculty of Science,
University of Duhok. The experimental work
was done in the Animal Biotechnology
Laboratory, Scientific Research Center, Faculty
of Science, University of Duhok. These animals
had free access of standard diets as pellets and
water.
These animals were randomly divided into
two groups (10 females for each group) as
follow: Group (1): Intraperitoneally (i. p.)
injected with 0.025 mg/kg (B.W.) cisplatin
(Kocak Farma, Uskudar, Istanbul, Turkey (as
single dose). Group (2) (control group):
Intraperitoneally injected (single dose) with
equivalent volume of normal saline (0.9%
NaCl).
Dissection and Histological Preparation
Ten females (5 from each group) were
dissected after 6 days and other ten females were
dissected after 12 days from injection. Animals'
body weight was measured before injection and
at the time of dissection (6 and 12 days). Thence
right and left ovaries were removed, weighted
and then fixed in 10% formalin for 48 hr for
histological and histopathological studies.
After fixation, the ovaries were processed for
paraffin wax embedding by dehydrating through
a scending concentration of ethanol and cleared
in xylene, infiltrated and is embedded in paraffin
wax. Serial ovarian tissue sections were cut at 6
µm thick using rotary microtome (KD-1508A,
China). Sections were stained with ordinary
Harries Hematoxylin and Eosin stains for further
investigation (Bancroft et al., 1999).
The ovarian follicular toxicity was evaluated
by examination of morphological changes. Then
the number of healthy and damaged follicles
(Primordial follicles, unilaminar primary follicle,
multilaminar follicle, antral follicle, Graafian
follicle and corpus luteum) was estimated.
Statistical Analysis:
Results were presented as the mean ± SE.
Significance of differences between means of
groups was evaluated using ANOVA.
Differences were considered significant when p<
0.05.
Results
Effect of Cisplatin on Body Weight
As shown in table (1), treatment with 0.025
mg/kg (B.W.) cisplatin for 12 days caused no
significant changes in the body weight (P< 0.05)
of the treated group compared with their counter
part of control group.
Effect of Cisplatin on Ovarian Weight
No significant effect of cisplatin was
observed on ovarian weight (P< 0.05) compared
with the control group (table 1).
Effect of Cisplatin on Morphologically
Normal Ovarian Follicles Number
The number of morphologically normal
follicles in each group was calculated, so, the
statistical analysis showed the following:
1. Primordial Follicles:
As shown in table (2), no significant
difference in primordial follicle number was
observed after 6 days compared with that of 12
days after cisplatin treatment. But the statistical
analysis showed that treatment with cisplatin
caused highly significant decreased in the
number of morphologically normal primordial
follicles at (P< 0.05) in the group of 6 days (2.68
± 0.43) compared with control group of 6 days
(13.08 ± 0.98, and in treated group of 12 days
(3.80 ± 0.59) compared with control group
(13.20 ± 0.93).
2. Unilaminar Follicles (Primary Follicles):
The number of morphologically normal
unilaminar follicles was significantly decreased
at (P<0.05) in group of (12 days after cisplatin
treatment) (0.60 ± 0.12) compared with the
group of 6 days (1.72 ± 0. 21). In addition, these
follicles showed a significant decreased number
among both cisplatin treated groups (6 and 12
days) compared with the mentioned periods of
control group s at P<0.05 (Table 2).
246
Generally, the control group showed a
significant increased number of these follicles
after 12 days compared with that of 6 days.
3.
Multilaminar
Follicles
(Secondary
Follicles):
The number of the morphologically normal
multilaminar
follicles
was
decreased
significantly in cisplatin treated group of 12 days
compared with group of (6 days) (0.12 ± 0.08,
1.40 ± 0.30) respectively (Table 2). The same
table also showed that treatment with cisplatin
caused significant decreased in the number of
multilaminar follicles in both groups (6 and 12
days) compared with the same periods of control
groups.
4. Antral Follicles:
Morphologically normal antral follicles in
cisplatin treated group of 12 days after treatment
was seen completely absent in this study (0.00 ±
0.00) at P<0.05. While in group of 6 days after
cisplatin treatment the number decreased
significantly (2.0 ± 0.47) compared with their
number in control group of (12 days) (4.76 ±
0.53).
Cisplatin treated group (6 days after
treatment) also showed significant decreased in
the number of this follicle compared with control
group (6 days) Table (2).
5. Graafian Follicles:
Cisplatin treatment resulted in damage to the
ovarian
tissue
and
the
number
of
morphologically normal graafian follicles was
null in 12 days of treatment (0.00 ± 0.00)
compared with their number in group 6 days
after cisplatin treatment (0.80 ± 0.16), but the
difference was significantly compared with the
control group (1.20 ± 0.24 (Table 2).
6. Corpus Luteum:
No significant differences in the number of
normal corpus luteum between both control and
cisplatin treated group was obsereved at P< 0.05
(Table (2).
Percentage of Morphologically Damaged
Ovarian Follicles in Cisplatin Treated Groups
Table (3) shows that the effect of cisplatin on
the ovarian follicles was increased with time
after treatment. Therefore the percentag of
247
morphologically damaged ovarian follicles
(Unilaminar primary follicle, multilaminar
primary follicle, antral follicle and Graafin
follicle) was increased after 12 days from initial
treatment with 0.025 mg/kg (B.W.) cisplatin.
But the percentage of damage in primordial
follicle was decreased with time after treatment
and this value was 73% in group of 6 days after
treatment while in group of 12 days after
treatment this value became 67%.
Histological and Histopathological Studies
The histological studies of ovarian tissues in
control group showed normal histological
appearance (Fig. 1 A and B).
Treatment with cisplatin resulted in damage
to ovarian tissues, and this harmful effect was
increased with time after administration. So, the
most pronounced histopathlogical changes were
observed in both cortical ovarian follicles and
medullary layer. These changes included
detachment of tunica albugina from the ovarian
tissue which observed in both cisplatin treated
groups (Fig. 2 A and B); destruction of ovarian
tissues which associated with moderate
congested blood vessels (Fig. 2 C and D).
In general ovarian tissues of group 6 and 12
days after cisplatin treatment showed no healthy
primordial multilaminar primary follicles and
(antral and Graafin) follicles which appeared
with pyknotic and shrinked ova (Fig. 3 A and C)
(Fig. 4 E and F); the pyknosis of granulosa cells
and detachment of these cells from the basement
membrane in addition to the disorganization of
granulosa cells and disruption of intracellular
contacts among them and oocytes in primordial,
unilaminar, multilaminar, antral and graafian
follicles (Fig. 3 A, B, C, D, E and F) (Fig. 4 A,
B, C, E and F). As shown in Fig. (3 D),
treatment with cisplatin, caused several degrees
of degeneration in ovarian follicles which were
devoid of healthy ova were recorded), most of
Graafian follicles were observed without ova,
corona radiata and cumulus Oophorus,
deforming oocyte, vacuoles were observed in
side the follicles (Fig. 4 C) and destruction of
theca cells (Fig. 4 B and D).
Groups
Control
Treated
with cisplatin
(0.025 mg/ kg)
248
Table (1): Effect of cisplatin on the weight of body and ovary of the animals
Periods
(Mean ± SE)
After 6 day
After 12 day
After 6 day
Animal
No.
5
5
5
After 12 day
5
B. W. Before
B. W. After
Ovary W.
a
25.10±0.43
25.0±0.44a
25.14±0.67a
a
28.22±1.91
27.58±0.66a
25.86±1.60a
0.01±0.01a
0.00±0.00a
0.01±0.00a
25.16±0.80a
28.42±1.27a
0.01±0.00a
Journal of University of Zakho, Vol. 2(A), No.2, Pp. 245-255, 2014
Means with different letters within each column differed significantly P (ANOVA) < 0.05.
Table (2): Effect of cisplatin on the number of ovarian follicles
Groups
Control
Treated with
cisplatin (0.025
mg/kg)
Animal
No.
Primordial
Follicles
Unilaminar
Follicles
(Mean ± SE)
Multilaminar
Follicles
Antral
Follicles
Graafian
Follicles
Corpus
Luteum
6 day
5
13.08±0.98a
5.08±0.33b
6.84±0.38a
5.48±0.73a
0.72±0.15a
2.56±0.29a
After 12 day
After 6 day
5
5
13.20±0.93a
2.68±0.43b
8.72±0.61a
1.72±0.21c
7.52±0.35a
1.40±0.30b
4.76±0.53a
2.0±0.47b
1.20±0.24a
0.80±0.16a
2.60±0.43a
2.16±0.24a
After 12 day
5
3.80±0.59b
0.60±0.12d
0.12±0.08c
0.00±0.00c
0.00±0.00b
2.24±0.44a
Periods
After
Means with different letters within each column differed significantly P (ANOVA) < 0.05.
Table (3): The percentage (%) of morphologically damaged ovarian follicles in cisplatin treated groups:
Groups
6 days after cisplatin
treatment
12 days after cisplatin
treatment
Primordial
follicle
Unilaminar
primary
follicle
Multilaminar
primary follicle
Antral
follicle
Graafian
follicle
73
69
78
58
0
67
88
98
100
20
Figure (1): A and B: Photomicrographs of ovarian tissues in control group showing normal structure of ovary
which surrounded by tunica albugina (arrow). Note the presence of normal unilaminar and multilaminar primary
follicles (double head arrow) in (Fig. A); and antral follicle (double head arrow) in (Fig. B). (X 80)
Figure (2): A: group of 6 days and B: group of 12 days after cisplatin treatment; these figures show the
detachment of tunica albugina (arrow) from the destruction ovarian tissue. C: group of 6 days and D: group of 12
days after cisplatin treatment, shows the destruction of ovarian tissues, note the presence of congested blood
vessels (arrow). (A and B: X 400) (C and D: X 80).
249
Figure (3): Morphological feature of degenerative changes in the ovarian follicles (group 6 days after cisplatin
treatment): (A): shows non healthy primordial follicles with condensed ovum (arrows), and pyknotic granulosa
cells in unilaminar primary follicle (double head arrow). (B): shows the detachment of granulose cells from the
basement membrane (double head arrow) and pyknotic granulose cells (arrows). (C): Multilaminar primary
follicle with shrinkage and pyknotic ovum (arrow), in addition to the disorganized granulose cells (double head
arrow). (D): shows several degree of degeneration in ovarian follicles which devoid of healthy ova. (E): this
figure shows mature follicle (double head arrow), but the ovum, corona radiata and cumulus Oophorus are
absent in addition to damaged follicles (arrows). (F) Disorganization of pyknotic granulose cells in antral
follicles (star). (A,B,C and F: X 400) (D and E: X 80).
250
Figure (4): Morphological feature of degenerative changes in the ovarian follicles (group 12 days after cisplatin
treatment): A: Detachment of granulosa cells from the basement membrane (arrow) and disruptions of their
intracellular contacts among them and oocytes. B: The deforming oocyte (arrow) and disorganization of pyknotic
granulosa cells associated with disruption of intercellular contacts among them. Note the destruction of theca
cells (star). C: Degenerating multilaminar primary follicle which appeared with pyknotic granulosa cells (double
head arrow). Note the presence of vacuoles (arrows) in side the follicles and the destruction of theca cells (star).
D: The deforming oocyte in unilaminar primary follicle (arrow) and destruction of surrounding tissue (star). E:
The shrinkage and degeneration of ovum (arrow), disorganization and disruption of pyknotic granulose cells in
antral and Graafin follicles in (E and F respectively). (X 400 )
251
Discussion
Cisplatin is an antineoplastic agent that is
used commonly in human and veterinary
medicine to treat a variety of malignant tumors.
But in the doses used for antitumor therapy,
cisplatin is a very toxic substance that caused
many adverse effects to the tissues (Arbaje et al.,
1993; Malarczyk et al., 2003). This toxicity was
observed and recorded in the results of present
study, which showed that treatment with 0.025
mg/kg (B.W.) cisplatin as a single dose caused
damaged to ovarian tissues resulted in
significant decreased in the number of ovarian
follicles and this effect was increased with time
after administration. This effect was observed
and recorded also by many authors such as
Türkyılmaz et al., (2008), they showed in their
study that single dose of 5 mg\Kg cisplatin
caused significant decrease in number of rats
primordial, primary, secondary and tertiary
follicles. Altuner et al., (2013) also showed that
treatment with cisplatin caused infertility due to
ovarian toxicity and reduce the number of
primordial follicles which constitute a
considerable portion of ovarian reserve.
Chemotherapeutic agents as indicated by
Meirow et al., (1999) not only destroying the
tumor tissues, but also cause damage over
multiplying germinal epithelium of cells such as
ovary. Whereas the damage inflicted by
cytotoxic agents on rapidly multiplying cells
such as included in bone marrow and thymus, is
reversible; the damage induced over primordial
follicles which do not have the ability to
regenerate, is of progressive and irreversible
character. Thus, damage on ovarian follicles
inevitably leads to premature ovarian
insufficiency and infertility. Therefore from this
result
it's
appeared
that
reproductive
performance is not important in determining
ovarian damage taking place following
chemotherapy but the most important parameter
is known to be the number of primordial follicles
forming a significant portion of the follicular
pool.
Cisplatin is one of the first generation
platinium compounds and is commonly used for
treatment of ovarian, breast, testicular, and
bladder cancers. Cisplatin induces cytotoxicity
by interference with transcription and/or DNA
replication mechanisms
(Florea and Büsselberg, 2011). That’s mean
binds covalently to guanine and adenine on
DNA and thus prevents transcription and DNA
replication (Gerschenso and Paik, 2001). At the
same time, exhibits anti-tumoral activity by
increasing toxic influence and apoptosis as a
result of binding with nuclear and cytoplasmic
proteins within the cell (Gustavo et al., 2002).
But the mechanism of action of cisplatin toxicity
on ovaries has not been explained thoroughly.
However, it is thought that increased production
of free oxygen radicals and decreased production
of antioxidants have an impact on the occurrence
of cisplatin toxicity (Weijl et al., 1998; Chirino
and Pedraza-Chaverri, 2009).
It has been
claimed that organ damage related to free
radicals occurs as a consequence of disrupted
antioxidant defense mechanisms (Altuner et al.,
2013).
cisplatin induces cytotoxicity, e.g., by
interference with transcription and/or DNA
replication mechanisms. Additionally, cisplatin
damages tumors via induction of apoptosis,
mediated by the activation of various signal
transduction pathways, including calcium
signaling, death receptor signaling, and the
activation of mitochondrial pathways.
The cytotoxic effect of cisplatin not
concerned with ovarian follicles but also include
grnulosa cells. Therefore the results of the
present histopathological study of ovarian tissue
showed that cisplatin caused sever damaged to
granulosa cells and this damaged was increased
with time of treatment. Himelstein-Braw et al.,
(1976) have been proposed to explain the
cytotoxicity occuring after chemotherapy. Most
important of those is known to be apoptosis of
oocytes. Alkylator agents show their cytotoxic
affects by causing an irreversible damage on
granulosa cells which are known to be the most
important component of developing follicles,
particularly primordial follicles.
Generally treatment with antitumor drugs
such as cisplatin, mitomycin C, etoposide and
cyclophosphamide induce apoptosis in male and
female germ cells which resulted in the
destruction of male and female gonads
(Nakagawa et al., 1997; Vaisheva et al., 2007;
Waheed and Esa, 2012; Altuner et al., 2013). In
conclusion: treatment with single dose of
cisplatin caused damage and significant decrease
in number of ovarian follicles which resulted in
females' infertility.
252
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NEW RECORDS OF FUNGI ON WHEAT GRAINS FROM IRAQ
Samir Khalaf Abdullah1 and Halben Ismat Mohammad Atroshi2
Biology Department, Faculty of Science, University of Zakho, Kurdistan Region – Iraq.
2
Department of Plant Protection, Faculty of Agriculture and Forestry,University of Duhok, Kurdistan Region - Iraq.
1
ABSTRACT
The present study recorded eight fungal species on wheat grains for the first time in Iraq.
These included Arthrinium phaeospermum, Bipolaris sorokiniana, B.spicifera, Chaetomium
elatum, Emericella rugulosa, Eurotium herbariorum Nigrospora state of Khuskia oryzae and
Ulocladium alternariae. Brief descriptions with photographs are provided for the newly recorded
species.
INTRODUCTION
F
ungi associated with seeds are
responsible for both pre-and postemergence damping-off of grains thus causing a
reduction in germination. They can also causing
mycotoxicoses in live-stock, poultry and humans
(Agrawal and Sinclair, 1987). Seed –borne
pathogens may also causes seed abortion, seed
rot, seed necrosis and production of metabolites
(such as toxins) which may alter grain
composition or metabolism or render it unfit for
human or animal consumption (Christensen and
Kaufman, 1969, 1974; Naraiah et al.,1986).
Moreover, close association with seeds
facilitates the long term survival, introduction
into a new areas and widespread dissemination
of pathogens (Agrawal and Sinclair, 1987).
Therefore, the study of seed-borne fungi is
very important to determine the health of grains
and to protect them from seed-borne pathogens
(HGCA, 2012).
During our continuous survey on seed-borne
fungi from different economic crops growing in
Iraq (Abdullah and kadhum, 1987; Abdullah and
Al-Mosawi, 2006, 2009, 2010; Haleem et
al.,2013), several interesting fungal species
newly recorded on wheat grains from Iraq have
been identified and briefly described along with
photographs.
aestivum L.) and twelve samples of durum wheat
(T. durum Desf.).
Detection of seed-borne fungi
Fifty grains were taken randomly from each
sample (total of 1300 grains) and were surface
disinfected with 1% sodium hypochlorite for 5
minutes, then washed twice with sterilized
distilled water. The seeds were dried on
sterilized filter paper and placed on two media
(Potato dextrose agar (PDA) (Himedia
laboratories,India) and Oat meal agar (OTA):30
g oat (Quiker oat),15 g agar 1L water in five
replicates for each medium, each replicate
contains 10 grains . Chloramphenicol (50 mg/L)
was added to each medium to inhibit bacterial
growth. Plates were incubated at 25°C for 6-10
days under near ultraviolet (NUV) light at 12
hours interval of alternation with darkness
(Mathur and kongsdal, 2003). Pure cultures from
growing colonies were obtained by transferring
fungal colonies individually on different fresh
media
plates
(MEA
(Himedia
laboratories,India),PDA
and
OTA)
for
identification.
Fungal Identification
All species identifications were according
to the keys and descriptions provided by Ellis
(1971, 1976); Arx et al.1986; Sivanesan, 1987;
Klich(2002); Watanabe(2002) and Guarro et
al.,(2012).
Sample sources
Twenty six samples of wheat grains were
obtained from official sources in Duhok provine
(Department of field crops, Faculty of
Agriculture and Forestry,Duhok University and
from directorate of agricultural research,Duhok)
and from silos at Shikhan and Zakho. Fourteen
samples were belonging to soft wheat (Triticum
RESULTS AND DISCUSSION
Five mitosporic fungi and three teleomorphic
ascomycetes have been identified, briefly
described and discussed as below:
Arthrinium
phaeosprmum
(Corda)M.B.Ellis
Mycol.Pap.103:8(1965). Figure 1.
256
Colonies on PDA are at first hyaline,
becoming brown to dark brown when conidia are
produced, reaching 44 mm diam in 2 weeks at
25˚C. Mycelium are mostly superficial,
septate.Conidiophores
are
micronematous,unbranched,subhyline to pale
brown , varying in length up to 60 um long and
1-5 um wide. Conidiogenous cells are discrete,
5-9×3-5 um. Conidia are often found in tight
clusters along a narrow conidiophore,
lenticellular, and smooth, pale brown to brown,
9-11×5-7um in size with longitudinal germ slit.
Specimens examined:
The species is very common on both soft
wheat and durum wheat grains. Representative
dried and living cultures have been deposited at
mycology
bank,
Plant
Protection
Department,Faculty of Agriculture and Forestry,
Duhok university.
This is the first report for the species in
Iraq. However, the species has been reported
from wheat grains in Scotland (Flannigan, 1970)
and from freshly harvested wheat from
Argentina (Broggi et al., 2007).An Arthrinium
sp was also reported common on soft white
winter wheat grains from Ontario, Canada (Clear
and Patrick, 1993). The species was also
detected from spelt wheat (T.spelta L.) grains in
Poland (Kurowski and Wysocka, 2009).
Figure 1: Arthrinium phaeosprmum conidia and conidiophores.
Scale bar = 10 um.
Bipolaris sorokiniana (Sacc. ) Shoem . Can .
J. Bot . 37: 884 (1959)
Teleomorph :
Cochliobolus sativus ( Ito& kurib ) Drechsler
ex Daster . Ind .J . Agric . Sci . 12: 733 (1942).
Figure 2
Colonies on PDA are dark brown growing
rapidly reaching 65 mm in 2 weeks at 25 ˚C.
Mycelium in forming a velvety layer, dark
brown, smooth on veruculose .Conidiophores are
erect, pale to dark brown up to 200 um long and
6-10 um wide and bearing 1-6 conidia at short
distances in the upper half. Conidia are curved to
straight smooth , olivaceos brown , fusoid to
broadly ellipsoidal, terminal partly each cell
subhyaline, 3-12(mostly 6-10) septate, 40120×20-28um.
257
Specimen examined: The species has been
isolated in one occasion for each of soft wheat
and durum wheat; dried and living cultures have
been deposited at mycology bank, at Plant
Protection Department, Faculty of Agriculture
and Forestry, Duhok University.
This is the first record for the species on
wheat grains in Iraq. However , the fungus was
recently isolated from a naturally infected roots
of a winter wheat
plants collected from
commercial field in Diwaniya governorate ,
middle Iraq ( Sarhan , 2013 ).
Bipolaris spicifera ( Bainier ) Subram ,
Hyphomycetes 756 (1971 ).
Teleomoraph : Cochliobolus
spicifer
Nelson , Mycologia 56 : 198 (1964 ). Figure 3
Colonies on PDA are dark brown to black ,
rapidly growing reaching 70 mm and 2 weeks at
25 C˚ .Conidiophores are pale to med brown
with obvious and numerous scars , up to 250 um
long at 4-8 um wide . Conidia are straight,
cylindrical, rounded at ends, smooth, 3- septate,
golden brown, 20-38 × 9 - 13 um.
detected in one occasion from durum wheat.
Dried and living cultures have been deposited at
mycology bank, at Plant Protection Department,
Faculty of Agriculture and Forestry, Duhok
University.
This is the first report for the species on
wheat grain in Iraq. However, the species was
previously detected from wheat grain important
to Iraq from Hungary and India (Juber and ALSalahi , 2006 ).
Figure 2: Bipolaris sorokiniana conidia. Scale bar= 25 um.
Figure 3: Bipolaris spicifera conidia. Scale bar= 10 um.
258
Chaetomium elatum
Kunze,Mycol.Hefte.1:16(1817). Figure 4 (A, B).
Colonies on PDA are white with pale
aerial mycelium reaching 60 mm diam in 2
weeks at 25 ˚C and often with yellow exudates.
Ascomata are spherical or ovate maturing within
2 weeks with brown wall, ostiolate 170-350um
size. Ascomata hairs are numerous, long,
dichotomously branched mainly in the upper
part, dark brown, regulose or warty, septate,45um thick at base . Asci are clavate, 8-spored,
evanescnt, 30-40×14-18 um. Ascospores are
liminiform, bilaterally flattened, brown at
maturity 8-11×7-9 um with an apical germ pore.
Specimen examined: The species is very
common on both soft wheat and durum wheat
grains. Representative dried and living cultures
have been deposited at mycology bank at Plant
Protection Department, Faculty of Agriculture
and
Forestry,
Duhok
University.
This is the first report for the species on wheat
grains in Iraq. However, the fungus was
previously reported from maize(zea mays L.)
grains and sunflower (Helianthus annuas ) seeds
in Iraq (Abddullah and Al-Mousawi,2006,2010)
,from leaves of sugarcane (Sacharum
officinarum L.) cultivars in Iraq (Abdullah and
Saleh,2010) and from soil at date palm
plantations and from surface sediments of Shatt
Al-Arab river and its creeks in Basrah, Iraq
(Abdullah
and
Zora,1993,Abbullah
and
Abbas,2008).
A
B
Figure 4: Chaetomium elatum. A: Ascomata with dichotomously branched hairs. Scale bar = 20um.
Asci and ascospores. Scale bar =10um.
259
. B:
Emericella rugulosa (Thom&Raper) E.R.Beng.
Mycologia 47:680 (1955). Figure 5 (A, B).
Anamorph
:Aspergillus
rugulovavus
Samson&W.Gams. in Samson and Pitt (eds.)
Advances in Penicillium and Aspergillus
systematics ,New York .49.1984.
Colonies on MEA are green reaching 55 mm
diam. in 2 weeks at 25˚C with light yellow
reverse. Cleistothecia are abundant developed
within 2 weeks, 250-300 um diameter. Hule cells
are globose, 18-20 um in diameter. Ascospores
are grayish red, 3.5-4.5×3-3.5 um with rugulose
convex wall and two equatorial crests. Conidial
heads are short columnar. Conidiophores are
smooth, 60-80×3-4 um. Vesicles are ovate to
flask-shaped 8-12 um wide, biserriate, strigmata
4-7×3-3.5 um . philides are 6-7.5um long and 33.5um wide. Conidia are green in mass,
spherical to subspherical 2.5-3.5 um diam.
detected in one occasions for each of soft and
durum wheat. Dried and living culture has been
deposited at mycology bank, Department of
Plant Protection, Faculty of Agriculture and
Forestry, Duhok University.
This is the first report for the species on
wheat grains. However, the species was
previously reported from soil in Kurdistan
region, Iraq (Abdullah and Abdullah, 2009;
Abdullah and Saadullah, 2013).
A
B
Figure 5. Emericella rugulosa. A: Asci and ascospores. Scale bar =5um.
anamorph. Scale bar = 10um.
.
.B: Hule cells and Aspergillus
260
Eurotiom
herbariorum
(WH.Wigg.)
Link.Mag.Gesell.Naturf.Freunde,Berlin
3:31(1809). Figure 6 (A- C).
Anamorph: Aspergillus glaucus (L.)Link .Mag,
Gesell.Naturf.Frunde, Berlin 3:82(1809).
Colonies on MEA are cream to orange buff
reaching 50mm diam. in 2 weeks at 25˚C
.Cleistothecia are abundant, 100-160um diam,
yellow to orange yellow encrusted with red
hyphae. Peridium composed of a single layer of
thin-walled pseudoparenchymatous cells of
A
B
261
texture angulrta. Asci are 8-spored, globose to
sub globose 11-14 um in diam. Ascospores are
white to yellow, 6-7×5-5.5um with pronounced
furrow and 2 equatorial crests with convex
surface finely roughened.Conidial heads sparse,
radiate to loosely columnar, pale blue green with
clavate to ellipsoidal uniseriate vesicle, 15-35um
in diameter.Conidia are globose to ellipsoidal,
dull green, thick walled, spinose to spinulose 58mm in diameter.
C
Figure 6: Eurotiom herbariorum. A: Aspergillus anamorph. Scale bar =5um. B: Cleistothecia. Scale bar = 50
um. C: Asci and ascospores. Scale bar =5um.
This is the first report of the species from
wheat grains in Iraq. However, the species has
been recently isolated from soil at grapevine
plantations in Duhok (Abdullah and Saadullah,
2013).
Nigrospora state of Khuskia oryzae Hudson.
Trans.Br.mycol.Soc.46:355-360(1963). Figure 7.
Anamorph : Nigrospora oryzae (Berk.&Br.)
Petch, J.Indian Bot.Soc.4:24(1924).
Colonies on MEA are black reaching 40mm
diam. in 2 weeks at 25˚C.Aerial mycelium is
wooly and hyphae are up to 9mm wide.
Conidiophores are short pale brown, bearing
conidia singly and terminally. Conidia are black,
globose in end view and ellipsoidal in side view,
measuring 13-17 um diam.(mostly 12-14um).
Specimen examined: The fungus has been
found in one occasion on durum wheat. Dried
and living cultures have been deposited at
mycology bank; plant protection Department,
Faculty of Agriculture and Forestry, Duhok
University.
This is the first record for the species on
wheat grains in Iraq. However, Juber and AlSalahi(2006) reported a Nigrospora sp. on wheat
grains imported to Iraq.
262
Figure 7: Nigrospora state of Khuskia oryzae conidia and hyphae. Scale bar = 10 um.
.
Ulocladium alternariae (Cooke) Simmons, Mycolgia 59:82-83(1967). Figure 8.
Colonies on PDA are reaching a diameter of 50 mm in 2 weeks at 25˚C.Mycelium is pale brown,
smooth 4-5 um. Conidiophores are golden brown up to 100×4-7um. Conidia are golden brown,
smooth, obvoid to broadly ellipsoidal, 20-30×12.5-18 um, with (1)-3-5 transverse septa and 1 or 2
longitudinal or oblige septa.
Specimen examined: The species has been isolated from both soft wheat and durum wheat grains
during this study. Representative dried and living cultures have been deposited at mycology bank at
Plant Protection Department, Faculty of Agriculture and Forestry, Duhok University.
This is the first record for the species in Iraq. However, the species has been reported on wheat
grains collected from Kerman province, Iran (Gohari et al .,2007).
Figure 8. Ulocladium alternariae conidia. Scale bar =10 um.
263
.
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from Ontario ,Canada. Can. Plant Dis .Surv.
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M. B. (1971). Dematiaceous Hyphomycetes,
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England 608 pp..
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Br. Mycol. Soc..55:267-276.
Gohari , A. M, sedachat, N. , Javan - Nikhah, M and Sabeririseh, R. (2007). Mycoflora of wheat grains in the
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(2012). Atlas of Soil Ascomycetes. CBS
Biodiversity series 10, CBS –KNAW Fungal
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and Waesi, I. M. (2013). Fungi associated with
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Agriculture and Horticulture Department Board.
HGCA publication, Warwickshire, U.K.
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associated with wheat grains imported to Iraq. Iraqi
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Species, CBS, Utrecht, The Netherlands, 116pp.
Kurowski,t.p.and Wysocka, U. (2009). Fungi
colonizing grains of winter spelt grown under two
production systems. Phytopathologia 54:45-52.
Mathur, S. B and Kongsdal, O (2003).Common laboratort
seed health testing methods for detecting fungi.
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production in wheat flour and its effect on protein
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A.R.T.(2013)
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Curvularia, Drechslera,Exserohilum and their
teleomorphs. Mycological papers No.158, C.A.B.
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Fungi. CRC Press, London,UK.486 pp.
264
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265
PATHOGENIC MICROORGANISMS ASSOCIATED WITH DIARRHEA IN
INFANTS AND CHILDREN IN DUHOK PROVINCE, KURDISTAN REGION /
IRAQ”
Awaz H. H. Badry, Ahmed Y. Jameel, Wijdan M. S. Mero
Dept. of Biology, Faculty of Science, University of Zakho, Zakho, Kurdistan Region-Iraq.
Abstract
This study was performed to assess the incidence of microorganisms causing diarrhea in infants and
children of both sexes and different ages from 12 days to 14 years in Duhok province, during the period
from October 2012 to June 2013, in which 522 diarrheal samples (278 males and 244 females) were
collected from infants and children attended pediatrics Heevi teaching hospital in Duhok city coming from
different regions of Duhok province. Out of 522 samples 426 (81.61%) were positive for bacteria, followed
by 146(27.97%) parasites, 69(13.21%) viruses and 5 (0.96%) fungi. The most prevalent enteric pathogens
were Escherichia coli 305(58.43%), followed by Entamoeba histolytica 134(25.67%), Klebseilla spp.
105(20.1%), and Rotavirus 57(10.91%). Mixed infections were documented in 129 (24.71%) cases, with
the maximum being with bacteria and parasite in 62(11.87%). The most frequent microorganisms
encountered in mixed infections were E. coli and E. histolytica at rates of 64.34 and 62.01%, respectively.
Infants less than 2 years showed the highest rate (72.22%), while children aged 12-14 years were least
infected (0.96%). Moreover, the rate of infection in males was greater than females, but statistically this
difference was nonsignificant.
Keywords: Microbial Diarrhea, Bacteria, Parasites,Viruses, Infants and Children
Introduction
Gastroenteritis is the most common cause of
morbidity and mortality in children worldwide
(Revelas, 2012). Although diarrhea kills about 4
million people in developing countries each
year, it remains a problem in developed
countries as well; diarrhea is common in all age
groups but is more common in infants, annually
at least 1500 million episodes of diarrhea occur
in children under age of 5 years (Khan et al.,
2004).The risk of children in this age group
dying from diarrheal disease is 600 times greater
in developing countries than in developed
countries (Vandepitte et al., 2003).The
etiological agents of diarrhea include a wide
range of viruses, bacteria and parasites (Bueris et
al., 2007). They are transmitted by ingesting
contaminated food or drink, by direct person-to
person contact, or from contaminated hands.
Human hands usually harbors microorganisms
both as a part of person’s normal microbial flora
as well as transient microbes acquired from the
environment (Tambekar et al., 2009).
This disorder is much more serious in infants
than in older children, and this is mainly due to
the relatively more marked disturbances in
fluids, electrolytes and acid-base balance
produced in infants (Brimble and Barltrop,
1984). In developing countries, summer
outbreaks of diarrheal diseases are largely due to
bacterial agents; this has been reported in
Turkey, Saudi Arabia and Egypt (Al-Sekait,
1998 and National Guideline, 2001). While
amoebiasis is primarily a tropical disease, but it
is more closely related to sanitary and
socioeconomic conditions than to climate
(Gendrel et al., 2003).
Very limited information are available on
diarrheal cases in children in Duhok province,
therefore, the aim of this study was to perform a
survey and to identify the causative agents of
diarrhea in infants and children of both sexes
and different ages.
Materials and Methods
Sample collection
In this study 522 diarrheic stool samples were
collected from infants and children of both sexes
and different ages ranged from 12 days to 14
years, who attended pediatrics Heevi teaching
hospital in Duhok city coming from different
regions of Duhok province from October 2012 to
June, 2013. After collection, if it was not
possible to deliver the sample to the laboratory
within 2 hours of its collection, a small amount
of the fecal specimen (together with mucus,
blood and epithelial threads, if present) was
266
collected on 2 to 3 swabs and placed in a
container with transport medium (Cary–Blair),
and taken to the laboratory.
All culture media used in the study were
prepared according to the procedures that
recommended by the manufacturing companies.
Sample processing
In the laboratory the collected samples were
processed following the standard laboratory
protocol (WHO protocol) in the Public Health
Research laboratories, as
1 Macroscopical examination
The color, consistency, presence of blood and
mucus and any other abnormalities were
observed macroscopically and documented.
2 Microscopical examinations
This was performed by 2 methods:
2.1. Direct wet mount method
A small fleck of the specimen was placed in a
drop of diluted lugal's iodine with normal saline
on the center of a clean slide mixed thoroughly
by a wooden stick, then covered with a cover
slide and examined by microscope, firstly with
10x then 40x. To look for parasites, pus,
leukocytes etc. From each sample 3 slides from
different parts of the sample were examined,
detected microorganisms were recorded.
267
2.2 Concentration technique (Zinc sulphate
floatation) (Faust et al., 1938)
This method was used to detect protozoan
cysts, helminthes ova and larva, in which about
2gm from each stool sample was mixed with 1012ml of normal saline. The mixture was strained
through two layers of wet surgical gauze, and
centrifuged for two minutes at 1500-2000 rpm.
The supernatant fluid was decanted and the
sediment was resuspended in normal saline and
centrifuged again, this process was repeated for
three times. A centrifuge tube was filled with
zinc sulfate close to the rim and covered with a
cover slide and centrifuged again at 2500 r.p.m
for one minute. The cover slide was transferred
to a slide containing one drop of lugal's iodine,
then was examined under 10x,40x and 100x.
The detected organisms were recorded.
Bacteriological isolation (cultivation)
After delivering stool samples to the
laboratory for bacteriological examination and
characterization, the following steps as indicated
in the flow chart were followed:
Identification of viruses
Viruses were identified using cer test
Rotavirus- Adenovirus Card test, approximately
100 mg of stool specimen was transferred by a
stick into the stool collection tube with diluent
samples, and then the tube was shacked in order
to assure good sample dispersion. After that 4
drops of solution provided with the kit were
added into the circular window marked with an
arrow, finally the results were read at 10 minutes
by observing the coloring bands:
Negative: Only one green band appeared
across the central window in the site marked
with letter C (control line).
Rotavirus positive: In addition to the green
control band, a red band (Rotavirus test line)
also was appeared in the site marked with the
letter T.
Adenovirus positive: in addition to the green
control band, a blue band (Adenovirus test line)
was appeared in the site marked with the letter
T.
Rotavirus-Adenovirus positive: all the lines
were appeared in the result region.
Isolation and identification of fungi:
After gram staining, the yeast isolates were
subcultured on candida chrome agar for the
identification of candida species, and incubated
at 35 ◦C for 24-48 hours, to permit the
development of colored colonies. The
presumptive identification was made by color
and morphology of the colonies; the isolates
were further identified microscopically and
morphologically.
Statistical analysis
Statistical analysis was carried out using
Graph Pad Prism 5 program. The chi-square test
was used and P < 0.05 was considered to be
statistically significant (Sokal and Rohlf, 2009).
Results and Discussion
1. The prevalence of microorganisms in the
diarrheic samples
The distribution of enteric microorganisms in
the examined diarrheal samples is shown in
tables (1 and 2). As it is obvious from the tables
that all of the examined samples (522) were
infected with various types of microorganisms,
some with more than one species of
microorganisms,
the
highest
percentage
(81.61%) of infection was with bacteria,
followed by parasites (27.97%), viruses
(13.22%) and the lowest was with fungi
(0.96%).
The isolated bacteria included, E. coli (at
highest rate, 58.43%), followed by K.
spp.(20.1%), Shigella and Pseudomonas spp.(2.3
and 0.77%), the recorded parasites were E.
histolytica at highest rate(25.67%), G.
lamblia(2.11%) and only one case of H.nana,
the viruses included Rotavirus at highest
rate(10.92%) and Adenovirus(2.3%), regarding
fungi only 5(0.96%) cases of candida spp. were
recorded. Furthermore, it is worthwhile to
mention that 24.71% of the examined samples
showed mixed infection as indicated in table (3).
A comparable prevalence of bacteria (87%)
among children was recorded by Kilic et al
(2007) in Gaziantep (Turkey). While, AlKhateeb (2008), recorded a higher bacterial
prevalence (95%) in Kut city.
However; a lower prevalence rates of
bacterial infection ranged between 33 to 44.84%
were recorded among children and infants in
various parts of Iraq, such as AI-Qadisiya,
Baghdad, Tikret and Karkuk Governorate a rate
of 46.52% was recorded by Esmaeel et al.
(2009); Ali et al. (2009); Mahmoud, (2010), and
Ibrahim (2012). The rate of parasites in this
study was comparable to that found among
children in Baghdad (25.56%) (Ibrahim, 2012).
On the other hand, a lower rate (11.6%) than that
reported in the present study was detected
among children in Kerbala (Hasan, 2010).
While, Mahmud (2009) and Hadi (2011)
recorded much higher rates (34.6 % and 43.1%)
with parasite infection among children in AlSowera city and Thi-Qar, respectively. The viral
figures in the present study was very close to
that found among children (14.42%) in Shiraz
(Iran), while in other Iranian cities Tabriz,
Mashhad and Tehran lower rates of viral
infection were recorded, which were, 7.56, 7.76
and 8.97%, respectively (Jadali et al., 2012). On
the other hand, Hussan (2012) in Baghdad
reported a much higher incidence of viral
infection (42.4%) among children. Regarding
fungi only 5 cases were detected.
Diarrhea is caused by a wide variety of
microorganisms including bacteria, parasites,
viruses, and fungi. But in the present study it
appeared that the main causative agents were
bacteria and parasites as they were encountered
in the highest rates among the studied samples
this may be due to poor hygienic condition of the
children and the atmosphere that they live in, as
268
most cases of diarrhea are transmitted via the
fecal-oral route through a variety of agents,
including contaminated food and drink, person
to person, hand to mouth contact, contact with
contaminated objects and possibly flies (Nguyen
et al.,2006, and Ali et al., 2009)
Table (1): The distribution of various microorganisms in the examined Diarrheic stool samples (n=522)
Types of Microorganisms
Bacteria
Parasites
Viruses
Fungi
No. infected
%
No.
infected
%
No. infected
%
No. infected
%
426
81.61
146
27.97
69
13.21
5
0.96
Table (2):; Types of microorganisms recorded in diarrheic samples.
Microrganismis
1. Bacteria
E. coli
Klebsiella spp.
Shigella spp.
Pseudomonas spp.
Total
2. Parasites
E. histolytica
G. lamblia
H.nana
Total
3.Viruses
Rotavirus
Adenovirus
Total
4. Fungi
Candida albicans
Total
2. Mixed infections
Table (3) shows the frequency of mixed
infections encountered in diarrheal samples. The
mixed infections with two or more
microorganisms was documented in 129
(24.71%) of the examined samples. The
correlation between bacteria and parasites was
the most common, and found in 62 (48.06%) of
total mixed cases. However a lower rates
(15.5%) of mixed infections ranged from 11 to
15.5% were recorded among children in
Northern Jordan, Saudia Arabia, Turkey, and
Burkina Faso (Youssef et al., 2000; El-Sheikh
and El-Assouli, 2001; Turhanoglu et al.,2012,
and Bonkoungou et al., 2013). E. coli and E.
histolytica were the most frequent pathogens,
269
Number
infected
%
of infection
305
105
12
4
426
58.43
20.12
2.30
0.77
81.6
134
11
1
164
25.67
2.11
0.20
31.42
57
12
69
10.92
2.30
13.22
5
5
0.96
0.96
occurring at rates of 64.34 and 62.01%,
respectively in mixed infection cases. The high
occurrence of E. histolytica might be attributed
to the fact that the cysts of E. histolytica, are
resistant to chlorination, they are killed by
heating only and also the unsanitary practice
associated with child living environment (e.g
playing in contaminated dirt and water, sucking
dirty fingers and other objects, etc.) (Nguyenet
al., 2006; Raddam and Hasson, 2008).
Among the bacteria-parasite mixed infection
E. coli+ E. histolytica was the leading agents
appeared in 36(27.90%) of total mixed
infections. While in Tikrit the most common
type of mixed infection among children was
found to be bacteria-bacteria which were
identified in 64.3% of the total mixed infection
cases (Alrifai et al., 2009). Moreover, E. coli
was also appeared to be an important pathogen
in bacteria-virus infection. E. coli+ rotavirus
recorded in 16(12.40%) cases. However, a lower
incidence of mixed infection E. coli+ rotavirus
(1.15%) was recorded among children in Kirkuk
(Zaman et al., 2012), while Klebsiella
spp.+rotavirus recorded in 6(4.65%) cases. A
lower rate of mixed infection E. histolytica
+rotavirus (2.4%) was obtained from children in
Kirkuk (Zaman et al., 2012).
Table (3): The frequency of mixed infections among 522 cases of diarrhea
Microorganisms recorded in mixed infections
1. Bacteria + Parasite
E.coli + Entamoeba histolytica
Klebsiella spp. + Entamoeba histolytica
Shiglla spp. + Entamoeba histolytica
E.coli + Giardia lamblia
E.coli + Klibsiella spp.+ Entamoeba histolytica
Total
2. Bacteria + Virus
E. coli + Rotavirus
E. coli + Adenovirus
Klebsiella spp. + Rotavirus
Total
3. Bacteria + Bactria
E. coli + Klebsiella spp.
E.coli + Pseudomonas spp.
Total
4. Parasite + Virus
Entamoeba histolytica + Rotavirus
Entamoeba histolytica + Adenovirus
Total
5. Parasite + Parasite
Entamoeba histolytica + Giardia lamblia
Entamoeba histolytica + Hymenolepsis nana
Total
6. Bacteria + Parasite + Virus
E. coli+ Entamoeba histolytica + Rotavirus
E. coli+ Entamoeba histolytica + Adenovirus
Total
7. Parasite + Fungus
Entamoeba histolytica + Candida albicans
8 Bacteria + Fungi
E. coli + Candida albicans
Total number of cases
Frequency
%
36
15
7
3
1
62
Frequency
16
5
6
27
Frequency
17
1
18
Frequency
12
1
13
Frequency
2
1
3
Frequency
2
1
3
Frequency
2
Frequency
1
129
27.90
11.62
5.42
2.32
0.77
48.06
%
12.40
3.87
4.65
20.93
%
13.17
0.77
13.95
%
9.30
0.77
10.07
%
1.55
0.77
2.32
%
1.55
0.77
2.32
%
1.55
%
0.77
24.71
3. The distribution of microorganisms according to the age
The distribution of enteric microorganisms in
different age groups is shown in table (4). The
highest percentage of infection was in the age
group from 12 days to 2 years which was
66.28% (346) with bacteria, 13.79 %( 72)
parasites and 10.53 %( 55) viruses. This
outcome could be due to exogenous factors such
as reduction of breastfeeding along with increase
in food supplementation in the second year of
life. The supplementary food can become
contaminated in the preparation process under
poor hygienic conditions, therefore, continuing
breastfeeding of children and maintaining
personal hygiene by those who prepare food for
children can also play an important role in
reducing the incidence of diarrhea (Arif and
Naheed, 2012). Similarly Rabatti and Rasheed
(2008) in Erbil reported the highest rate of
infection with different microorganism in this
age group, in Baghdad, Ibrahim (2012) also
recorded the highest prevalence of bacteria,
parasites, viruses and fungi in children less than
2 years (61.88% for 1 day to 1year and 21.52%
for 1.1 to 2 years) than other age groups. While
270
to record the lowest rates of infection with
different microorganisms in the age group 12-14
years as the rates of bacteria and parasites were
0.57 % for each of them, 0.38% for viruses, and
fungi were not detected in this group. Statistical
analysis of the results showed the presence of
significant relation between the age and the rate
of infection for bacteria and parasites (p<0.05),
while the relation was non-significant relating to
viral and fungal infections (p>0.05).This could
be due to the differences in hygiene practices of
the populations, environmental and host factors
(Huruy et al., 2011).
The present results partly agree with those of
Hasan (2010) as he found a similar relationship
between parasite infection and child age in
Kerbala. Sule et al. (2011) also confirmed a
similar correlation for bacteria and age
distribution in Kaduna (Nigeria). Moreover,
Forbes et al. (2001), in a study carried out in
Western Australia, also found that the fungus
(Candida) is not associated with child age
(Forbes et al., 2001). While Motamedifar et al.
(2013), found a significant relation between viral
infection and age among children in Shiraz, Iran.
Bonkoungou et al (2013) found that the bacterial
and viral infection were most common (76%)
among children less than 2 years in Burkina
Faso. Moreover, the majority of the bacterial
infections were found in children less than 2
years in a study conducted by Cajetan et al.
(2010) in Abuja (Nigeria). On the other hand,
Hasan (2012) recorded lower prevalence of
parasites in Kerbala among children less than 2
years (7.14%) than children from 2-4 years
(38.5%).
A dramatical decrease of all microorganisms
occurred at the age <2-4 years, the rate of
bacteria dropped to 7.08% (37), parasites 5.36%
(28), viruses 0.76% (4) and fungi 0.38(2). At the
age above 4 to 6 years the decrease of the rates
of microorganisms continued significantly, in
this age group the rate of bacteria became
2.29%(12)
parasites
2.49%(13),
viruses
0.38%(2) and fungi 0.9% (1). At the age of 6-8 a
slightly higher rate (2.87) of bacteria was
recorded, while the rates of parasites, viruses and
fungi declined to become 2.29%, 0.19% and 0%,
respectively. From the ages 8-10 years to 12-14,
the microorganism figures fall but more steadily
Table (4): The distribution of enteric microorganisms in different age groups (No=522)
Age
groups
(years)
Less than
2
<2-4
<4-6
<6-8
<8-10
<10-12
<12-14
Total
Types of pathogens
Bacteria + ve
Parasites + ve
Fungi +ve
Frequency
%
Frequency
%
Frequency
%
Frequency
%
346
66.29
72
13.80
55
10.54
2
0.39
37
12
15
8
5
3
426
7.09
2.30
2.87
1.54
0.95
0.58
81.61
28
13
12
10
8
3
146
5.37
2.49
2.29
1.92
1.53
0.58
27.97
4
2
1
2
3
2
69
0.77
0.39
0.19
0.39
0.57
0.39
13.22
2
1
0
0
0
0
5
0.39
0.20
0
0
0
0
0.96
P-Value<0.05
<0.05
4. Relationship between gender and age for
diarrhea
The relationship between different age
groups, gender and rate of diarrhea is shown in
table (5). The overall rate of diarrhea showed
that more males were having diarrhea as
compared to females. Among the total enrolled
cases 278 (53.26) males and 244(46.74%)
females were diarrheic over the all age groups as
shown in table (5). but this difference between
both sexes was statistically non-significant (p>
o.o5). This agrees with other studies conducted
by Kolahi et al., (2008) in Tehran (Iran), Johargy
271
Viruses + ve
>0.05
>0.0
et al.(2010) in Makkah (Saudi Arabia),Yilgwan
and Okolo (2012) in Nigeria. This may be due to
the fact that both sexes have the same chance of
exposures to the environmental conditions and
contaminated sources of infection such as food
and water. While some other studies reported
higher rate of diarrhea in male children (Nguyen
et al., 2006, Ansari et al., 2012 and Arif and
Naheed, 2012) in Hanoi (Vietnam), Kathmandu
(Nepal) and rural area (Pakistan), respectively.
Regarding the age, the highest rate of
diarrhea was among the ages from few months
to less than 2 years (table.5).The greater risk of
diarrhea in the first 2 years of life is due to
combined effects of declining levels of
maternally acquired antibodies, the lack of active
immunity in the infant, the introduction of food
that may be contaminated with fecal bacteria and
direct contact with human or animals feces when
the infant start to grow. Most enteric pathogens
stimulate at least partial immunity against
repeated infection or illness, which helps to
explain the declining incidence of disease in
older children (Sule et al., 2011). A rapid
decrease in the rates of diarrhea occurred in both
males and females at the age group 4-6 years,
the rate of diarrhea in males became 3.64% and
in female 3.07%. This rapid decline in the rates
of diarrhea continued to the age 6-8 years to
become 1.53% for males and 2.30% for females.
On the other hand, the lowest rate of diarrhea
was among the age group 12-14 years, since
only 0.96% of the children found with diarrhea.
These results disagree with some studies which
stated that there is no significant difference in
the occurrence of diarrheal illness in the general
population and school children in Nepal (Rai et
al, 1986; 1995; 2001; 2002; Ishiyama et al,
2001). Similarly Adhikari et al.(1986) and Ono
et al.(2001) also did not observe any significant
difference in the prevalence of enteric parasites
in children and adults as they indicated that both
children and adults, irrespective of sex, were
equally exposed to enteric parasites, particularly
diarrheagenic protozoa, and attributed it to
unplanned urbanization, which results in poor
sanitary and hygienic conditions, and
contamination of drinking water with fecal
matter.
Table (5): Relationship between gender and age groups for diarrhea
Gender
Age groups
(years)
Male
Total
Female
Less than 2
2-4
4-6
6-8
8-10
10-12
12-14
Number
201
34
19
8
7
5
4
%
38.50
6.51
3.64
1.53
1.34
0.96
0.77
Number
176
25
16
12
6
8
1
%
33.72
4.79
3.07
2.30
1.15
1.53
0.19
Number
377
59
35
20
13
13
5
%
72.22
11.30
6.70
3.83
2.49
2.49
0.96
Total
278
53.26
244
46.74
522
100
P>0.05
From this study we can conclude that most
cases of diarrhea were found to be associated
with bacteria (81.61%), with the highest
incidence of E. coli (58.43%), followed by
Klebsiella spp.(20.12%), Shiglla spp. (2.3%) and
Pseudomonas spp.(0.77%). Parasites were the
second leading pathogens, they contributed to
31.42% of the total cases, with the highest rate
being for E. histolytica (25.67), followed by G.
lamblia (2.11%) and only one case of H. nana
(0.76%). Viruses were recorded at a rate of
13.22%., the highest rate was with rotavirus
(10.92%) and 2.30% with adenovirus. With
respect to fungi, only 5(.96%) cases of Candida
albicans were recorded. Mixed infections with 2
or more microorganisms were documented in
129 cases, with the maximum being with
bacteria and parasites. The rate of infection with
different types of microorganisms was found to
be age dependent and gender independent.
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274
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275
HISTOLOGICAL CHANGES IN SOME ORGANS OF THE FEMALE RATS INFECTED
WITH TOXOPLASMA GONDII PARASITE ISOLATED FROM EMBRYO OF ABORTED
SHEEP
Liqaa H. Al-Delami1, Buthaina Hatim. Al-Sabawi2, FirasM.Basheer1, Hafidh I. Al-Sadi3
1
Department of biology, Education College for Girls, University of Mosul, Iraq.
2
Department of anatomy, College of medicine, University of Mosul, Iraq.
3
Department of pathology, College of Veterinary, University of Mosul, Iraq.
Abstract
The current study included identifying the lesions and the histological changes caused by the
intracellular parasite Toxoplasma gondii, which was isolated from embryos of aborted ewes.
Intraperitonially injected in 15 female albino rats (3 months old) with suspension containing 100 tissue
cysts
Histological section showed after four months of injection a chronic infection characterized by
autolysis in all sections, where the liver sections showed expansion in the central hepatic veins, congestion
in sinusoidal curves with irregularity in hepatic cords, and the presence of parasites in the liver cells. The
brain tissue showed vacuoles in the neurons with an increase in the number of Purkenji cells. Kidney
sections were characterized by degenerative and necrotic changes in the endothelial cells of the proximal
and distal convoluted tubules with Sloughing of necrotic and degenerated cells of the tubes which
accumulated inside the cavity. The parasites appeared in the endothelial cells of the glomerular tufts.
Ovary and the uterus showed increased vascular wall thickness, furthermore, the spleen showed autolytic
changes, deposition of pigment with the presence of parasites within the cells.
Keywords: Toxoplasma gondii, histopathological changes, Sheep.
Introduction
Toxoplasmosis, caused by Toxoplasma
gondii, is an economically important disease of
livestock, especially sheep and goats, as it can
cause early embryonic death, resorption, fetal
death, mummification, abortion, stillbirth, and
neonatal death.
Cats are the main reservoir for the
toxoplasmosis and they can contaminate the
environments of other animals and humans by
passing oocysts with their feces (Mohammed et
al., 2010).
Toxoplasma. gondii is an obligate
intracellular parasite that infects a wide variety
of hosts, including humans. Infection generally
occurs through the ingestion of either sporulated
oocysts shed in cat feces or viable tissue-cysts in
undercooked meat (Copprin, 2003; Barakat et
al., 2009). In addition, primary infection during
pregnancy
results
from
transplacental
transmission of tachyzoites which can result in
severe congenital disease in the fetus with
potential abortion (Copprin et al., 2003), causing
severe birth defects, such as hydrocephaly,
calcification,
neurological
defects
and
choriorentinits (Wong, 1994).
During acute infection, tachyzoites multiply
rapidly. They can invade and proliferate in all
nucleated cells by active penetration and form
parasitophorous vacuoles. After repeated
replication, host cells are disrupted and
tachyzoites disseminate via the bloodstream and
can invade many tissues, including the central
nervous system, eye, skeletal, heart muscles and
placenta. Replication leads to cell death and
rapid invasion of neighboring cells. The
tachyzoite stage causes a strong inflammatory
response and tissue destruction and, therefore,
causes clinical manifestations of the disease
(Montoya, 2004).
The
cell-mediated
immune
response
convered the tachyzoites into bradyzoites and
forms tissue cyst. These tissue cysts remain
viable and are capable of persisting for the life of
the host (Mordue et al., 2001; Montoya, 2004).
The severity of toxoplasmosis varies according
to the immune status of the individual, parasite
strain, and host species. In mammalian species,
it has been severe lesions of acute toxoplasmosis
have been observed in visceral organs such as
the liver, the lungs and the spleen. Some
epidemiological studies have reported an
association of Toxoplasma gondii infection with
liver cirrhosis (Hasan et al., 2013).
The purposes of the present study were to
observe the pathological changes occurring due
to the experimental infection of T.gondii in rats.
276
Material and Methods
In this study, female rats were used, which
are obtained from the animal house of college of
veterinary medicine, university of Mosul, after
breeding until giving birth to small babies, 15 of
these babies were selected for experiments, the
breeding took place under laboratory conditions
in the light cycle which was divided into 14-hour
of darkness and 10 hours of light ,at a
temperature of 22±2o C), the rats were supplied
with water and food daily until they became
adult to be infected intraperitonially with a
suspension containing 100 tissue cysts of T.
gondii /rat. The T. gondii tissue cysts were
obtained from sections of brain, liver, kidney,
spleen and lung tissue of aborted ewes. Tissue
infections were confirmed by the presence of the
parasite cysts in tissue sections of infected ewes.
In order to obtain the tissue cysts, these organs
were cut into small pieces using scissors and
forceps then digested in acid pepsin solution
(2.6gm pepsin enzyme, 5gm sodium chloride, 7
ml concentrated hydrochloric acid and 500
distilled water) by adding 1gm of tissue to 10 ml
of solution, then homogenized by a blender
using the maximum speed (10000 r/min), the
mixture was incubated at a temperature of 37 oC
for a period of 90 minutes, then strained through
several layers of sterile gauze, the yield was
centrifuged at a speeds of 400r/min for 10
minutes, the sediment was mixed with phosphate
buffer saline (pH 7.2-7.4) recentrifuged, then the
sediment was suspended in phosphate buffer
saline containing antibiotics (1000 IU of
penicillin and 10 microgram of streptomycin
/1ml) of phosphate buffer saline. About 0.4 ml
of this suspension which supposed to contain
100 tissue cysts of T.gondii was injected
intraperitonially to each rat aged 3 months
(Sukthana et al., 2003) .The infected rats were
kept in the laboratory for 4 months to develop
chronic infection. Then the animals were
scarified and the liver, brain, kidney, spleen and
ovary were removed and embedded in paraffin
wax, sectioned at 5-6µm stained with
hematoxylin eosin stain (H&E) (Pearse, 1985).
The sections were examined under light
microscope and photographed.
Result and Discussion
The microscopical examination of the
histological section of the infected liver tissues
(Figures.1 and 2) showed an expansion in the
central hepatic veins and disarrangement of the
277
hepatic cords with evidence of parasites in the
liver cells, Kupffer cells and sinusoids.
Brain tissue sections showed the occurrence
of vacuoles in the neurons with an increase in
the number of Purkenji cells( Fig.3).
Kidney sections were characterized by
degenerative and necrotic changes in the
endothelial cells of the proximal and distal
convoluted tubules of the kidney with sloughing
of necrotic and degenerated endothelial cells of
the tubes which were collected inside the cavity,
the parasites accumulated in the endothelial cells
lining the tubes and endothelial cells of the
glomerular tufts (Fig. 4 and 5). These changes in
infected tissues may be attributed to the
virulence of the strain, to the persistent parasite
antigens or their metabolic by products which
leads to the occurrence of necrosis (Laug et al.,
2006; Djurkovic-Djakovic et al., 2006; Ramzam
et al., 2009).
Ovary and uterus showed increase in the
thickness of the blood vessels walls (Fig.6),
while the spleen showed autolytic changes,
pigment deposits, with the presence of parasites
in different places of the spleen (Fig.7).
Infection acquired in embryos of aborted
lamb through transplacental transmission of T.
gondii from mothers infected during early stage
of pregnancy. The mother acquire the infection
through different modalities, the most important
of them is the process of dissemination of
infection through contaminated food with the
T.gondii tissue cysts(Aitken, 2007;Ahmed et al.,
2008).
The results showed that the histopathological
changes of the liver tissues might be due to the
fact that T.gondii interfere with the function of
mitochondria and shifting it to anaerobic
methods of energy production which is sufficient
for the work of sodium pumps, causing low
protein production and damaging the cell
membranes as well as the mechanism of
phagocytosis which occurs in the liver tissue
leading to necrosis in the tissue, this might be
the reason for an increase in the stimulation of
free radicals leading to the severity of the
histological effects in the liver (Sukhana et al.,
2003; Riyadh, 2005).
The histopathological changes in the brain
tissue which was demonstrated by occurrence of
vacuoles in nervous cells and the increase in the
number of Purkenji cells as the brain is the most
of the body tissues affected by T. gondii,
because the brain is rich in fatty materials and
this may be the reason for the preference of T.
gondii, the brain tissue as they need energy for
their existence and reproduction which causes
the most severe congenital malformations in the
brain (Buxton et al., 2007), as well as, brain
tissue is characterized by a lack of specific
immune defenses like antibodies, but it posses
non-specific immune defenses as microglial cells
(Sukhana et al., 2003; Buxton et al.,2007;
Williams et al., 2008).
The dissolution and degenerative changes in
the endothelial cells of the proximal and distal
convoluted tubules with the emergence of
degeneration and hemolytic cells as well as the
occurrence
of
the
renal
glomerular
inflammation, in addition,
to cellular and
vascular changes, may be due to the occurrence
of necrosis in the convoluted proximal and
distal tubules due to the thrombosis which
blocked the
blood vessels causing
immunological damage and degeneration,
followed by necrosis and desquamation of the
cells, this is called tubular necrosis. The
inflammation of the renal tubules and the
glomeruli may be due to the damage from the
immune mechanisms, and this is the most
important damage occurring to the basal
membranes or glomerular deposition of the
immune complexes inside the glomeruli causing
inflammation
associated
with
chronic
glomerulus infection (Sukthana et al., 2003;
Rodger et al., 2006).
The histological changes in the spleen may be
due to the effect of some enzymes such as acid
hydrolases which exudes form lysosomes and
increase leaching.
In case of ischemia any shortages or lack of
oxygen cause interruption of blood processing.
The enzymes exuded from damaged cells change
the compositions of complex organic substances
to simple inorganic substances such as water,
hydrogen sulphate, carbon dioxide, and nitrates.
These changes in the cell are quiet similar to
those occurring in necrotic cell and are more
tougher leading to autolysis more rapidly in the
highly effective organs as the spleen and the
lymph nodes, the main function of these organs
is the defense mechanism as the spleen is the
largest storage of macrophagic cells and is rich
in lymphatic vessels, that is why great
decomposition occurs in this tissue (Sukthana et
al., 2003; Pinheiro et al., 2009).
The hyperplasia and thickening of the walls
of the blood vessels of the ovary caused by
chronic infection of T. gondii, are attributed to
the disruption in the architecture of the ovarian
tissue due to hormonal imbalance in function of
the ovary affecting the growth and maturation of
the ovarian (Graafian )follicles and the
disturbance in the secretion of ovarian hormones
produce lesions in ovarian tissues (Husein &
Kridli, 2003).
The histopathological changes occurred in
rats after 4 months of experimental infection
with T. gondii tissue cysts were very severe as
compared to many studies performed by
researchers in which infection periods were
shorter range between 4-8 weeks and they
attributed the severity of the resulted
histological changes to the severity of the
infection with T. gondii isolated from embryos,
as this strain is more virulent in some animal
species than in other, in addition some
histological lesions start sharp and ends quickly
and others start sharp and became chronic, or
there may be severe lesions during the chronic
phase and continue. Other researchers attributed
the severity of the histopathological changes to
the high ferocity of T. gondii isolated from
embryos of aborted sheep a discouraging
immunoreactive animals and suffers from
stressful situations that is why T. gondii are more
virulentand cause severe
histopathological
effects in the host tissue (Savio & Nieto, 1995;
Riyadh, 2005; Djurkovic-Djakovic, et al., 2006;
Laug et al., 2006).
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Figures
Fig. 1: Section from the liver of infected rat showing expansion in the central hepatic veins (H&E stained.400x)
a
b
Fig. 2: Section from the liver of infected rat.(a) showing congestion, granular curves.(b) T.gondii tissue cysts ( H&E
stained. 400x)
280
Fig. 3: Section from the brain of infected rat showing vacuoles in the neurons with an increase in Purkenji
cells(H&E stained. 400x).
Fig. 4: Section from the kidney of infected rat showing expansion and degenerative changes of the lumen of
glomeruli (H&E stained. 400x)
.
281
Fig. 5: Section from the kidney of infected rat showing degeneration and dissolution of the alveolar cells lining the
distal tubules convoluted (H&E stained.400x)
Fig. 6: Section from the ovary of infected rat showing hyperplasia in ovarian cells and increase in the thickness of
blood vessels (H&E stained.400x)
15
282
Fig. 7: Section from the spleen of infected rat showing autolytic changes in spleen cells(H&E stained.400x)
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284
INCIDENCE OF HUMAN SCABIES IN DUHOK PROVINCE, KURDISTAN
REGION/ IRAQ
Wijdan M.S. Mero1 and Haliz Khalid Hassan2
Department of Biology, Faculty of Science, University of Zakho. Kurdistan Region-Iraq.
2
Directorate of Education, Ministry of Education, Duhok province, Kurdistan Region-Iraq.
1
Abstract:
This study was conducted in Duhok city to evaluate the incidence of scabies among outpatients from
both sexes and different ages from one year to over 60 years visited the dermatology clinic in Azadi
Teaching Hospital from September 2012 to April 2013. Other factors like residency, educational levels,
number of family members, source of contact and clinical manifestations were also studied. Scabies was
diagnosed in 522 (5.5%) out of 9450. Higher rates (20.11% and 20.5%) of scabies were observed among
age groups 1-10 and 11-20 years, respectively, then the rate decreased with the increase in the age. Female
patients had higher frequency than males (53.06% and 46.94%, respectively). Moreover, the highest rate
(60.53%) was found among illiterates patients and decreased with the increase in the level of education.
The urban and rural residents showed high prevalence rates (37.16 and 34.86%) as compared to
suburban (27.98%). About half of the cases occurred from household contact (50.98%) and 34.67%
acquired from outside homes, 13.78% from unknown sources and only 0.57% from contact with
prisoners. The clinical findings, 91.57% of the patients suffered from itching that turned to secondary
bacterial infection, the disease was generalized in 53.06%, localized in 29.12% of the cases affecting hands,
fingers and legs, and in 17.82% of the cases affected their abdomen, back and under arms. According to
disease duration 31.42% of the patients seek medical attention during the first week of having the
symptoms, 24.90 % during the second week, 17.63% during the fourth week and 26.05 % delayed medical
consultation for more than a month. Regarding living conditions, 21.83% of the patients lived in a house
with less than five persons and high standard of living, while 78.17% of the patients lived in a very
crowded house with 6 to 18 persons.
Key words: Scabies, Incidence, Socioeconomic factors.
Introduction
S
cabies is a very contagious itching
condition of the skin caused by a tiny
mite called the human itch mite Sarcoptes
scabiei (Rozendaal, 1997).The adult mites enter
the skin creating serpinginous borrows in the
upper layer of the epidermis, the female mite
lays her eggs in the skin burrows. Scabies is a
major global health problem in some indigenous
communities inside the developed countries and
in the third world communities (Scheinfeld,
2004). The risk of severe outbreaks was high in
institutions (including nursing homes and
hospitals) and among socially disadvantaged
populations and immunocompromised hosts
(Roberts et al., 2005 and Chosidow, 2006).
Many people suffer from scabies infestation
at any time (Chosidow, 2006 and Muhammad
Zayyid et al., 2010). It occurs in both sexes, at
all ages, higher in rural areas than in cities and in
children than teenagers (Lydden, 2005). Living
in colonies, public places and prisons may
increase the infestation (Shamsaddini et al.,
2000). Scabies is usually transmitted by direct
285
skin-to-skin physical contact, other objects; such
as clothing, bedding, furniture might have come
in contact (Heukelbach and Feldmeier 2006).
Clinical diagnostic symptom is range from
intense itching, usually in the interdigital fold
and sides of the fingers, buttock, external
genitalia and wrists within incubation period
from 1 to 4 days to pruritic papules and impetigo
with some secondary bacterial in complicated
cases. Diagnosis of scabies infestation usually is
based on appearance and distribution of the rash
and confirmed diagnosis by identifying the mite
or mite eggs or fecal matter (Chosidow, 2006).
In recent years, scabies appears to have become
endemic in Iraq and documented by several
studies .This study is designed to find out the
epidemiological profile of scabies in Duhok
Governorate
among
patients
visiting
dermatology clinics in and its correlation with
their socioeconomic status together with
examination of stool samples from infected
persons to exclude any parasitic infection.
Subjects: This study was conducted during
September 2012 to April 2013; in which a total
number of 522 scabies cases (277 females and
245 males) which were Clinically diagnosed by
consultant dermatologists out of 9450 patients
visited the Dermatology Clinic of Azadi
Teaching General hospital for investigation and
treatment. The patient’s ages ranged from 1 year
to over 66 years, and they were inhabitants of
various parts of Duhok city and nearby villages.
An interview and a questionnaire were used for
each patient after taking permission from
consultant physicians which included: Data
about gender, age, level of education,
occupation, if child level of parent’s education,
number of family members, residency, economic
status, source of contact, the sign and symptoms
of the disease, its duration, type of treatment and
recurrence of the disease.
The results were based on the analysis of 522
patients from both sexes of different ages from
one year to over 66 years with confirmed
diagnosis of the presence of Sarcoptes scabies
mites or eggs by microscopic examination of the
skin scrubs taken from the infested body parts of
patients enrolled in this study. Furthermore, all
stool samples taken from these patients were free
from any parasitic infection as demonstrated by
microscopic examination. The incidence of
scabies was 5.5% (table.1) indicating a high
frequency of scabies among patients presented to
the dermatology clinic. Other studies in Iraq
reported rates of 52%, 1.9%, 3.3%, 1.2% and
2.7% of scabies among patients from the lower
socioeconomic classes, respectively (Samarai,
1995; Al- Al-Rubaiy, 2001; Murtada, 2001;
Alaa, 2002, and Mahmood, 2011).
It is obvious from table(1) that the age groups
1-10 and 11-20 years showed the highest rates of
infestation which were 20.11% and 20.5%, in
male and females respectively. The rate of the
infestation decreased with the increase in age up
to the age of 60 years, over this age the rate
become 3.44%. However, this is in accordance
with that reported by Sharma et al. (1984); ElOkbi et al. (1993) and Al-Shawa (2007), they
indicated that scabies is more common in ages
less than 10 years up to 19 years and this
prevalence may be due to overcrowding, poor
living conditions and the prolonged contact
among patients and their family members. This
result contradicts with Walton et al. (2004) as
they stated that the prevalence of scabies is not
affected by the age. In the current study, females
showed higher rate of scabies infestation than
males (53.06 versus 46.94%). Similar
observations have been reported in other studies
such as Kenawi et al. (1993); Golchai et al.
(2003); Ciftci et al. (2006), and Lassa et al.
(2011), and attributed it to the study design, the
possibility of more exposure to infestation as
results of the type of work performed by females
in addition to poor hygienic measures and the
house hold activities. On the other hand, the
present results disagree with Sharma et
al.(1984); Mustafa et al.( 1997; Arjomandzadeh
et al.(2001; Al-Shawa, (2007; Al-samarai,(
2009); Najem et al.( 2009); Muhammad Zayyid
et al.( 2010); Fakoorziba et al.( 2011) and
Ibrahim et al.( 2012).
Table (2) demonstrates that the highest rate
of infestation (60.53%) was among illiterates
Stool samples
From each scabietic patient a stool sample
was taken which was kept in a clean labeled and
closed container to be examined later for parasite
investigation.
Microscopic examinations of skin scrapings
The definitive diagnosis of scabies is based
on the identification of mites, eggs and eggshell
fragments, from skin scrapings (e.g., from
scabietic papules or from under the fingernails)
or by the detection of the mite at the end of its
burrow. One or two drops of mineral oil were
applied to the lesion, which was then scraped or
shaved, and the specimens were examined after
clearing in 10% KOH with a light microscope
under low power(Chosidow, 2006).
Microscopic examination of stool samples
The collected stool samples were examined
in the laboratory by direct wet mount as follows:
About 2 mg of stool was emulsified in a drop of
warm (37°C) saline and one drop of Lugals
iodine on a clean slide using a wooden stick on
an area of about 2 cm in diameter, then covered
with cover slip and examined under the
microscope. Using low power objective lens
(10X), suspected objects are examined using the
high objective lens (40X) to detect parasites. At
least 2 to 3 smear were examined for each
sample.
286
lack of health education which support the study
of Ciftci et al. (2006) who reported a significant
relationship between the rate of infestation and
education. In this aspect, the present results
disagree with those reported by Al-Chalabi
(2009) in which she stated that only 13.1% were
illiterates, 35.8% completed the primary level
and 16.9% were university graduates.
patients. The rate decreased with the increase of
the level of education except the slightly higher
rate (17.81%) for patients who completed
primary school probably due to sample size. The
high rate of illiterate may be due to the low
income, sleeping outside home, low standard of
living and poor hygienic condition or low
education and this is indication of poverty and
Table 1: Distribution of scabies according to age and gender
Age group
(Years)
Total No.
infested
%
infested
Male
number
%
infested
Female
number
%
infested
1- 10
105
20.11
67
12.83
38
7.27
11-20
21-30
31-40
107
89
91
20.5
17.05
17.43
59
42
33
11.3
8.05
6.32
48
47
58
9.2
9
11.11
41-50
73
14
25
4.8
48
9.2
51-60
39
7.47
10
1.92
29
5.56
More than 60
18
3.44
9
1.72
9
1.72
Total
522
100
245
46.94
277
53.06
Table 2: The distribution of scabies according to educational status.
Socio-demographic
characteristic
Total No of
infested
Total % of
infestation
No of
infested
males
% of
infested
No of
infested
females
% of
infested
98
18.77
218
41.76
Education
Illiterate
316
60.53
Can read and write
70
13.41
43
8.23
27
5.17
Completed primary school
Completed secondary
school
Higher education
93
17.81
51
9.77
42
8.04
39
7.48
19
3.63
20
3.84
4
0.77
3
0.59
1
0.2
Total
522
100
214
40.99
308
59.01
As shown in Table 3, urban and rural
residents have somewhat, higher rates of
infestation as compared to suburban residents,
which were 37.16%, 34.86% and 27.98%,
respectively. Other studies showed that scabies
is highly endemic in rural areas, for example, in
Bangladesh (Hayee et al., 1998), Egypt (Hegazy
et al., 1999) and in the UK (Downs et al., 1999).
In general, most of the people were residents of
overcrowded areas characterized by low income
in addition to low levels of education and
sanitation. Table (4) displays the clinical
287
findings among the studied patients. About half
of the cases had a household contact (50.98%) as
a possible source of scabies infestation, 34.67%
of the cases acquired the infestation from outside
their homes, 13.78% from unknown sources and
only 0.57% from prisoners. The high percent of
household contact might be due to the
overcrowding and general socioeconomic
level. On the other hand, 34.6% of the cases
acquired the infestation from outside their
homes. The patients claimed that they got the
Table 3: Distribution of scabies according to residency.
Residency
Total No
infested
Total % of
infestation
Urban
194
37.16
No of
infested
males
96
18.39
No of
infested
females
98
Rural
182
34.86
82
15.7
100
19.16
Sub- urban
Total
146
522
27.98
100
75
253
14.38
48.47
71
269
13.6
51.53
Infestation while they were admitted to
hospital for other illness, or after visiting parents
or relatives, all these factors will assist in the
spread of the disease. Only 0.6% of the patients
acquired scabies by contact with prisoners, and
% of
infested
% of
infested
18.77
this indicates that the prisons of Duhok province
are clean. This study disagrees with that reported
by Roodsari et al. (2007) in which they reported
that the prevalence of scabies in Ghezel Hesar
prison was 2.2% (31 cases) from 1404 prisoners.
Table (4): Distribution of scabies according to source of contact.
Clinical
finding
(Source of
contact)
Total No of
infested
Total % of
infestation
No of
infested
males
%
infested
No of
infested
females
% of
infested
females
House hold
266
50.98
128
24.52
138
26.46
Outside home
181
34.67
78
14.94
103
19.73
unknown
with prisoner
72
3
13.78
0.57
36
3
6.89
0.57
36
0
6.89
0
Total
522
100
245
46.92
277
53.08
disease was described as generalized in almost
half of the cases (53.06%), while a localized
form (hands, fingers and legs) of the disease was
documented in 29.12% of the cases. In the
remaining 17.82 of the cases, the diseases
affected their abdomen, back and under arm.
This disagree with what have been reported by
Kenawi et al. (1993) and Al-Chalabi (2009) as
they found the most affected sites were the
abdomen and back (100%) and Palms, wrists
and interdigital webs were involved in 72% of
the cases.
As shown in Table (5), 91.57% of the
infested patients suffered from itching which
leads to a secondary bacterial infection other
studies which carried out worldwide also
showed the same findings (Lawrence et al.,
2005). Both sexes nearly have the same rate of
itching which is intensified during night due to
increased mite burrowing activity, their
secretions and defecation (Arlian, 1989). Similar
result regarding itching have been reported by
Walker and Johntone (2000); Wendal and
Rompalo (2002) and Anne et al.(2007). The
Table 5: Distribution of scabies according to clinical symptom and site of infection
Clinical symptom
Total No of
infested
% of
infestation
No of
infested
males
% of
infested
No of
infested
females
% of
infested
Itching
478
91.57
227
43.47
251
48.09
Pain or discomfort
44
8.43
18
3.46
26
4.98
Total
522
100
245
46.93
277
53.07
Site of infestation
Generalized infestation
277
53.06
130
24.9
147
28.16
Hand, finger and leg
152
29.12
82
15.8
70
13.4
Abdomen, under arm and back
93
17.82
33
6.34
60
11.49
Total
522
100
245
46.95
277
53.05
288
is much higher than rates reported by AlChalabi, (2009) who reported infestation rates
only 13% of the cases were reinfested with
scabies for the second time. As shown in Table
(7), overcrowding in the residence place was an
important attribute for the patients with scabies.
Only a small proportion (21.83%) of them lived
in a house with less than five persons. This
condition
offers
better
socioeconomic
circumstances and higher standard of living,
78.17% of the studied cases lived in a very
crowded residence with a household size of 6 to
18 individual. The results of this study indicate
that scabies was more prevalent among patients
from families with 5-10 persons living in a
house with
As shown in Table (6), 31.42% of the
infested patients with scabies seek medical
attention during the first week of having the
symptoms, 24.90% during the second week,
17.63% of cases during the 4th week and 26.05%
delayed their medical consultation for more than
a month after the start of the symptoms. There is
no any study in this direction in order to
compare the results. A positive past attack of
scabies was reported in 113 (21.65%) of the
cases, however, most of them reported that they
could not afford the medications for the whole
family members since the drugs are not available
in public pharmacies and they cannot afford to
buy it (personal communication). On further
enquiry, many of them showed inappropriate
application of the drugs. The infection rate here
Table (6): Distribution of scabies cases according to disease duration.
Duration of
scabies
(weeks)
Total No of
infested
Total % of
infestation
No. of
infested
Males
% of
infested
Males
No of
infested
females
% of
infested
females
1
2
164
130
31.42
24.90
91
65
17.43
12.45
73
65
13.98
12.45
4
up to month
Total
92
136
522
17.63
26.05
100
43
57
256
8.24
10.92
49.04
49
79
266
9.38
15.15
50.96
few rooms. Similar results were reported from other developing countries (Hegazy et al.,
1999,Larrosa et al., 2003, and Al-Chalabi, 2009) where scabies was more prevalent among large
families with a high crowding index at night due to close contact and sharing of beds that increase the
transmission of the scabies mite. The present study revealed that families of scabies cases were often
of large size and a high crowding index. This could implicate close contact and the sharing of beds in
the transmission of the scabies mite.
Table (7): Distribution of scabies patients according to the number of family members
Number of
family
Members
Total No of
infested
% of
infestation
No of
infested
males
% infested
No of
infested
females
% infested
1-5
114
21.83
50
9.57
64
12.26
6-10
237
45.40
122
23.37
115
22.03
> 10
171
32.77
73
13.98
98
18.79
Total
522
100
245
46.94
277
53.06
The present study concluded that the incidence of scabies was high among the outpatients visited
the dermatology clinic in Azadi Teaching Hospital, particularly in female in the age group from 1--20
years, in illiterate patients then decreased with the increase in the level of education, also in patients
who lived in a very crowded residence high rates of scabies were recorded namely the resident of
urban and rural areas and more than 50% acquired the infestation from household contact. Itching was
the prominent clinical symptoms with secondary bacterial infection in some of them.
289
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292
MULTILOCUS SEQUENCE TYPING OF KLEBSIELLA PNEUMONIAE
PRODUCING EXTENDED SPECTRUM Β-LACTAMASES ISOLATED FROM
KURDISTAN REGION, IRAQ.
Haval Mohammed Khalid*, Jaladet M.S Jubrael** and Samira Younis Yousif*
*Department of Biology, Faculty of Science, University of Zakho, Kurdistan Region – Iraq.
** Scientific Research Center, Faculty of Science, University of Duhok, Kurdistan Region – Iraq.
Abstract:
This study was purposed to sequence analysis of ESBLs genotype of K pneumoniae using partial
sequence and Multilocus sequence typing (MLST). A total of 275 K. pneumoniae isolatesinvolved three
general hospitals in Duhok, Erbil, and Sulymania, from September 2010 to June 2011. The Minimum
Inhibitory Concentration (MIC) was measured by Phoenix system that confirmed 187 ESBL producing
isolates followed by the Double Disk Synergy Test (DDST). Then, 12 isolates were selectedaccording to
sample diversity, high resistancy to β-lactam and cephalosporins and harboring combination of three
genes (TEM, SHV, and CTX-M). Partial sequence analysis of TEM; showed two different genotypes
regarding blaTEM as 9 isolates (75%) from different samples (wound, sputum and blood) from three
provinces harbor TEM-1 gene and 3 isolates (25%) only from urine in three provinces harbor TEM-198
gene. SHV analysis revealed characterization of selected isolates into six different genotypes. The common
genotype was blaSHV-11 involved five isolates from sputum and blood in Erbil and Sulymania provinces,
and wound in Duhok province. Only one genotype as all 12 isolates (100%) from different samples and
different provinces was found harbored CTX-M-15 gene. Multilocus sequence typing (MLST) study
performed using seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB and tonB). A total of 8
different sequence types (STs) were identified;ST11 was dominant sequence type, accounting 41.6 %( 5
isolates) and was harboring combination of TEM-1, SHV-11 and CTX-15 genes.
Key words: Klebsiella pneumoniae, ESBL,MLST.
Introduction
Klebsiella pneumoniae is an opportunistic
pathogen responsible for a high proportion of
nosocomial infections and are dramatically
increasing resistant to multiple antimicrobial
agents owing to the production of extendedspectrum beta-lactamases (ESBL) which confer
high resistance to third and fourth generation
cephalosporins (Brisseet al., 2000;Paterson et
al., 2000; Paterson et al., 2004). Hospital
outbreaks of K. pneumoniae isolates are
frequent, and the inter-hospital dissemination of
resistant strains has been described previously
(Arletet al., 1994; Monnet et al., 1997).
Multilocus sequence typing (MLST) is a
nucleotide sequence-based method that is
adequate for characterizing the genetic
relationships among bacterial isolates (Maiden et
al., 1998; Enright and Spratt 1999;Feilet al.,
2004). It provides unambiguous and portable
data that allow the implementation of multiuser
international databases (Jolley and Maiden
2004). For our best knowledge,Multilocus
sequence typing has never been validated for the
epidemiology of K. pneumoniaestrains in our
area using the diversity of some housekeeping
293
genes (Paterson et al., 2004). For this reason,
this study was designated to develop an MLST
scheme for clinical isolates of K. pneumoniae
andits evaluation for molecular characterization
of nosocomial dissemination.
Bacterial Isolation
Two hundred and seventy five (275) clinical
isolates of K. pneumoniae werecollectedfrom
various clinical specimens (urine, blood, sputum,
and wound) from September 2010 to June 2011
included three general hospitals (Duhok, Erbil
and Sulymania) at Kurdistan region/Iraq. All
these isolates were characterized and identified
using different conventional bacteriological and
biochemical standard methods.
Antimicrobial Susceptibility and ESBLs Test
Using the Phoenix System
The antimicrobial susceptibility and the
ability to produce ESBLs of all 275 isolates were
tested by the Phoenix system (Beckton
Dickinson Diagnostic Systems, USA) using
Gram-negative susceptibility card (GNS).
Double Disk Synergy Test (DDST)
All ESBL producing isolates were tested by
Double Disk Synergy Test (DDST) with disks
containing cefotaxime (30 µg), ceftazidime (30
µg), and ceftriaxone (30 µg) whichwere placed
25 mm (centre to centre) from an Amoxicillinclavulanic acid disk ( 30 and 10 μg, respectively)
and incubated at 35°C overnight (Kaur et al.,
2013).
Multilocus sequence typing
Sevenhousekeeping genes were selected to
establish the MLST. The primers used for
amplification of the gene fragments are shown in
table (1). PCR amplifications were carried out
under the following conditions: Denaturation
step at 95°C for 4 min, followed by 30 cycles of
the following conditions: denaturation at 95°C
for 1 min, annealing at 60°C for each primer set
for 1 min, and extension at 72°C for 1 min. then
the final extension step at 72°C for 5 mints. The
PCR products were purified using a PCR
purification kit (Qiagen-Germany), according to
the manufacturer’s recommendations, and then
the products were sequenced on an ABI3130
DNA
sequencer
(Applied
Biosystems,
Singapore). MLST was performed as described
by
Diancourtet
al.
(2005)
(http://www.pasteur.fr/recherche/genopole/PF8/
mlst/). Briefly stated, PCR fragments of the
seven housekeeping genes rpoB, gapA,mdh, pgi,
phoE, infB and tonB were obtained from
chromosomal DNA and directly sequenced.
Allelic profiles and sequence types (STs) were
designatedatthewebsite
mlst/Kpneumoniae.html).
Table.1 primers used for the amplification of the house keeping genes.
Locus
rpoB
gapA
Putative function of gene
Primer sequence
Beta-subunit of RNA
polymerase B
Glyceraldehyde 3Phosphatedehydrogenase
F: GGC GAA ATG GCW GAG AAC CA
R: GAG TCT TCG AAG TTG TAA CC
F: TGA AAT ATG ACT CCA CTC ACG G
R: CTT CAG AAG CGG CTT TGA TGG CTT
F: CCC AAC TCG CTT CAG GTT CAG
R: CCG TTT TTC CCC AGC AGC AG
F: GAG AAA AAC CTG CCT GTA CTG CTG GC
R: CGC GCC ACG CTT TAT AGC GGT TAA T
F(seq): CTG CTG GCG CTG ATC GGC AT
R(seq): TTA TAG CGG TTA ATC AGG CCG T
F: ACC TAC CGC AAC ACC GAC TTC TTC GG
R: TGA TCA GAA CTG GTA GGT GAT
F: CTC GCT GCT GGA CTA TAT TCG
R: CGC TTT CAG CTC AAG AAC TTC
F(seq): ACT AAG GTT GCC TCC GGC GAA GC
F: CTT TAT ACC TCG GTA CAT CAG GTT
R: ATT CGC CGG CTG RGC RGA GAG
mdh
Malatedehydrogenase
pgi
Phosphoglucoseisomerase
phoE
Phosphoporine E
infB
Translation initiationfactor 2
tonB
Periplasmic energy
transducer
Size
(bp)
Temp
(°C)
501
50
450
60
447
50
432
50
420
50
318
50
414
45
Detection of bla genes sequencing of ESBLs genes
The blagenes related to ESBL enzymes were assayed by PCR with the corresponding primers for
the TEM, SHV, and CTX-M ESBL types that shown in table (2), as described previously by Yun-Tae
et al.(2006).To specify the subtype of the blagenes, the amplified PCR products were sequenced on
both strands, the amino acid sequences were deduced from the nucleotide sequences using the Mega
and BioEdit programs and they were compared with the database of the website (http:
//www.blast.ncbi.nlm.nih.gov).
294
Table.2 PCR amplification and sequencing of ESBLs genes
ESBLs
primer
Primer sequence
Size
(bp)
F: CGCCTGTGATTATCTCCCT
R: ATCGTTGTCAGAAGTAAGTTGG
TEM
F(Seq): CGCCTGTGATTATCTCCCT
R(Seq):
ATCGTTGTCAGAAGTAAGTTGG
1cycle of 5 min at 94°C:30 cycles of (30 sec
1080
F(Seq): CGCCTGTGATTATCTCCCT
R(Seq): CGAGTAGTCCACCAGATCC
861
F(Seq): CGCTGTTGTTAGGAAGTGTG
R(Seq): GGCTGGGTGAAGTAAGTGAC
Resulst and Discussion
Partial sequence analysis of TEM; showed
two different genotypes regarding blaTEM as 9
isolates (75%) from different samples (wound,
sputum and blood) from the three provinces
harbor TEM-1 gene and 3 isolates (25%) only
from urine samples of the three provinces harbor
TEM-198 gene. Results obtained from SHV
analysis revealed the characterization of the
selected isolates into six different genotypes.
The most abundant genotype was blaSHV-11
involved five isolates from sputum and blood in
Erbil and Sulymania provinces, and wound in
Duhok province. Only one genotype as all 12
isolates (100%) from different samples and
different provinces was found harbored CTX-M15 gene.
MLST using seven housekeeping genes
(gapA, infB, mdh, pgi, phoE, rpoB and tonB)
was performed on 12 ESBLs producing K.
pneumoniae selected isolates obtained from
different samples and different provinces
according to the protocol described
for
K.pneumoniaeMLST
website:
mlst/Kpneumoniae.html)
Results from table (3) revealed that eight
different sequence types (STs) were identified in
this study.
ST11 was the dominant sequence type
accounting for 41.6 % (5 isolates). All 5 isolates
having ST11 (allelic profile 3-3-1-1-1-1-4) were
295
72°C); cycle of 10 min at 72°C
at 94°C, 30 sec at 48°C, 1 min at 72°C);
cycle of 10 min at 72°C
F: CGCTGTTGTTAGGAAGTGTG
R: GGCTGGGTGAAGTAAGTGAC
CTX-M
at 94°C, 1 min 30 sec at 45°C, 1 min at
F: CGCCTGTGATTATCTCCCT
R: CGAGTAGTCCACCAGATCC
SHV
PCR cycles
551
at 94°C, 1 min at 58°C, 1 min at 72°C); cycle
of 10 min at 72°C
obtained from sputum and blood (Erbil and
Sulymania) and wound (Duhok) table (3). These
isolates were also found to have the same
genotype regarding ESBLs (TEM-1, SHV-11,
and CTX-M-15), While the other 7 isolates
belonged to 7 different sequence types
including; ST14, ST15, ST37, ST48, ST268,
ST413, and ST514.
In South Korea ST11 was also found to be
the most dominant type in ESBLs producing K.
pneumoniae isolates obtained from urine and
bacteremia (Koet al., 2010). In China ST11 was
also reported as the dominant clone of ESBLproducing K. pneumoniae (Qi et al., 2011).
ST11 has been also described in different
European countries such as Hungary, the
Netherlands, France and Spain (Damjanovaet
al., 2008; Oteoet al., 2009; Tothet al., 2010).
All eight STs obtained from the 12 different
isolates regarding ESBLs genotype were found
to be 100% CTX-M-15 and the isolates obtained
from wound, sputum and blood were 100%
TEM-1
ESBLs producing K. pneumoniae isolates
obtained from urine samples were found to have
TEM-198 (table.3).
These results probably could represent
regional dissemination of selected isolates of K.
pneumoniae among the three provinces. Similar
findings were recorded in the Midwestern USA
(Kitchelet al., 2009).
Table.3 MLST of 12 selected ESBL producing K. pneumoniae isolates obtained from different sources and different
provinces in Kurdistan region/Iraq
No. of Isolates
Source
Isolate Kp1
Urine
Province
Duhok
TEM
SHV
CTX-M
Allelic Profile
ST
TEM-198
SHV-125
CTX-M-15
2-9-2-1-13-1-16
37
TEM-1
SHV-11
CTX-M-15
3-3-1-1-1-1-4
11
Isolate Kp2
Wound
Isolate Kp3
Sputum
TEM-1
SHV-148
CTX-M-15
2-1-2-1-7-1-81
268
Isolate Kp4
Blood
TEM-1
SHV-12
CTX-M-15
2-5-2-2-7-1-10
48
Isolate Kp5
Urine
TEM-198
SHV-28
CTX-M-15
1-1-1-1-1-1-1
15
Isolate Kp6
Wound
TEM-1
SHV-63
CTX-M-15
2-1-1-1-8-1-9
514
Isolate Kp7
Sputum
TEM-1
SHV-11
CTX-M-15
3-3-1-1-1-1-4
11
Isolate Kp8
Blood
TEM-1
SHV-11
CTX-M-15
3-3-1-1-1-1-4
11
Isolate Kp9
Urine
TEM-198
SHV-148
CTX-M-15
2-6-1-3-8-1-113
413
Isolate Kp10
Wound
TEM-1
SHV-28
CTX-M-15
1-6-1-1-1-1-1
14
Isolate Kp11
Sputum
TEM-1
SHV-11
CTX-M-15
3-3-1-1-1-1-4
11
Isolate Kp12
Blood
TEM-1
SHV-11
CTX-M-15
3-3-1-1-1-1-4
11
Erbil
Sulymania
From this study it could be speculated that
ST11 to be a good colonizer to capture plasmids
as these isolates are easily transmitted between
patients (Qi et al., 2011). Results from table (3)
shows that ST15 is a single locus variant (infB)
of ST14. ST15 was detected in China in just one
hospital in Hangzhou and spreads regionally
(Qiet al., 2011).
The dissemination in both Europe and Asia
may indicate that ST11 is one of the pandemic
clones
although
there
are
limited
epidemiological data on the worldwide
distribution of K. pneumoniae (koet al., 2010).
In conclusion, these isolates are worrying due
to their rapid transmission by plasmid or
mobilization of genetic elements between
hospitalized patients causing sporadic spreading
and worse effects, these bacteria may not be
detected in routine antibiotic susceptibility
testing. We should therefore pay careful
attention should be paid to this problem from the
public health point of view through monitoring
closely and strict infection control measures
should be adopted to control nosocomial
infections.
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@béØþnàaì@ pbåîŠíjíÜbÑïÜaíánØý@ bnïjÜa@ pa†b›à@ ëb¤@ óïÜbÉÜa@ bénàìbÕàì@ pbåïÉÜa@ Š†b—à@ À@ æîbjnÜa@ ¶a@ bÕj @ ÒïÜa@ óÉaíÜa@ ïánØý
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@æàì@ŠaŠ†ýa@pbåïÈ@æà@pýÈ@HERUI@ SìTEM-1@ËíåÜa@ÚÝn¸ì@óqþrÜa@pbÅÐba@æàì@âÍÝjÜaì@Ê“ÕÜa@Lì‹§a@pýÈ@æà@ (EWUIY
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@o¸.CTX-M-.15 gene.ÂáåÜa@íèì@óÑÝnƒ¾a@pýÉÜa@À@a‡yaì@b¹@ôÈa@‡ÕÐ@ blaCTX-M@@ï§a@ÂáåÜa@bàa@NÛíè†@óÅÐb«
(gapA, infB, mdh, pgi, @ HHousekeeping genesI@ pbåïu@ Êj@ ßbáÉndi@ ï§a@ ÊÔí¾a@ †‡Énà@ pbÉibnm@ Âïáåm@ óaŠ†
@pýÈ@ U@ À@ ST-11@ íè@ ò†bï@ ‹rØýa@ ðÉibnnÜa@ ÂáåÜa@ çbØì, óåîbnà@ êïÉibnm@ Ãb¹a@ çb@ w÷bnåÜa@ p‹éÄaì@ phoE, rpoB, tonB)
TEM-1, SHV-11 and CTX-15@ï§a@ÂáåÜa@ÚÝn¸@Üaì@HETQNVI
298
DACTYLOGYRUS SCRJABINENSIS (MONOGENEA: DACTYLOGYRIDE):
FIRST OCCURRENCE ON THE GILLS OF CAPOETA TRUTTA FROM IRAQ
1
Younis Sabir Abdullah1 and Shamall Mohammad Amin A. Abdullah2
Department of Pathological Analysis, Sulaimani Polytechnique University, Sulaimani, Kurdistan Region - Iraq.
2
College of Agricultural, University of Salahaddin, Erbil, Kurdistan Region - Iraq.
Abstract:
The momogenean Dactylogyrus scrjabinensis Osmanov, 1958 was identified on the gills of the cyprinid
fish Capoeta trutta (Heckel, 1843) from Sirwan river, southeast of Sulaimani governorate, Kurdistan
region, during the period from April to August 2014, in the present study for the first time in Iraq. The
full details of description and measurements of this parasite are given. The prevalence and intensity of
infection were 3.94% and 3.66 respectively.
Keyword: Dactylogyrus scrjabinensis, Capoeta trutta, Sirwan river, Iraq.
Introduction
The importance of fish parasites is directly
related to the importance of fish that they affect
(Hoffman,
1967).
Monogenea
are
hermaphroditic, direct life cycle, generally
permanent ectoparasites on body surface or gills
of marine, brackishwater, or freshwater fishes,
attached by posterior adhesive organ (Margolis
and Kabata, 1984). The genus Dactylogyrus is
the largest helminthes genus, with more than 900
species and generally has high host specificity
(Neary et al., 2012). Most Dactylogyrus species
parasitize cyprinids although certain species are
adapted to the more advanced fish families.
Furthermore, commercial and aquaculture
exploitation of cyprinids caused examination of
these species for potential pathogens (Gibson et
al., 1996).
A major identifying characteristic of
monogenean
parasite
is
their
haptor
attachment, anchors, number of hooks,
copulatory organ, gonads and four eye spots.
These characters exhibit a range of intraspecific
genetic or phenetic variation (Harris 1988). The
first information on genus Dactylogyrus from
the Iraqian freshwater fish was given by Ali et
al. (1986), who recorded D. cornu from six
species of fishes from Diyala river, southeast of
Baghdad.
In the present study, we have described and
reported the occurrence of D. scrjabinensis from
the gills of cyprinid fish C. trutta in Sirwan
river, Kurdistan region, Iraq.
Study area: Sirwan basin situated between
latitudes of 350 05' and 350 10' and between
longitudes 450 50' and 460 05', it represents a
boundary of Iraq- Iran at the northern east of
Iraq. The basin bounded by Biyara watershed at
the north and west, by Darbandikhan lake from
the south and by Iran from the east. It’s located
within high folded and thrust zones, there were
many mountains and valleys in this area
especially in Hawraman area. Sirwan river
stream is a perennial main stream which supply
water to Darbandikhan lake. The main part of
the area of this basin lies inside Iran (Barzinji,
2013).
Sampling: A total of 76 Capoeta trutta were
collected from Sirwan river near Halabja city by
local fishermen using gill netting twice monthly
during the period from April to August 2014.
The fishes were placed in a container with the
local river water, and transferred immediately to
the laboratory as soon as possible and were
examined. The fishes were identified according
to Coad (2010).
In the laboratory, gills of fishes were placed
in a separate Petri dish with only small amount
of tap water. Pieces of gill filaments were tiered
by a fine needle. Worms (after leaving the gills)
were removed from the water by a small pipette
and placed on a slide, with a few drops of water.
They were covered with a cover slip with
glycerin-gelatin. A piece of melted glyceringelatin was dropped with cover slip onto the
worms. The cover slip was dried by a blotting
paper carefully, and the worms in glyceringelatin are cautiously thickened (Kritsky et al.,
2004). Smears from the skin were taken by slide
299
scraping and examined directly under the
microscope. The measurement of worms was
achieved by ocular micrometer. The parasite
identification and the terms used were as
recommended by Bykhovskay-Pavlovskay et al.
(1962); Gussev et al. (1993); Pugachev et al.
(2010). Photos were taken with Sony Optical
Steady Shot Digital camera model DSC-W570,
16.1 mega pixels.
The survey showed the occurrence of one
monogenean
belonging
to
the
genus
Dactylogyrus. The following is a brief account
on this parasite.
Dactylogyrus skrjabinensis Osmanov, 1958
(Fig. 1):
Host: Capoeta trutta (Heckel, 1843)
Site infection: Gill filaments
Prevalence of infection: 3.94%
Mean intensity of infection: 3.66
Locality: Sirwan river, southeast of Sulaimani
governorate, Kurdistan region, Iraq.
Description: worms small or medium. Total
length about 0.7-0.8 mm, width 0.10-0.15 mm.
Length of marginal hooks 0.030-0.035 mm.
Length of median hooks 0.050-0.060 mm, main
part 0.035- 0.040 mm, inner root 0.025-0.030
300
mm, outer root 0.005-0.008 mm, point 0.150.020 mm. Size of connecting bar 0.008-0.012 x
0.035-0.045 mm. Size of supplementary bar
0.030-0.034 x 0.050-0.054 mm. Total length of
copulatory organ 0.060-0.065 mm.
The description and measurement of the
present specimen are similar to those reported by
Bykhovskay-Pavlovskay et al. (1962) and
Pugachev et al. (2010) for D. skrjabinensis in
which found on gill filaments of Aral barbel
from Amu-Darya river in Russia and on gill
filaments of Luciobarbus brachycephalus from
Central
Asia
respectively.
Also,
this
monogenean was recorded on the gills of Barbus
barbulus and Capoeta capoeta from Armand
river, Chaharmahal va Bakhtyari province, Iran
(Raissy and Ansari, 2012).
This parasite was never been reported from
any fish species in Iraq before. Therefore, the
present parasite is considered as the first record
in Iraq.
By recording this species of Dactylogyrus (D.
scrjabinensis) in the present study, a total of 78
species of Dactylogyrus become known from
different species of fishes in Iraq. Among this
number, 48 species were recorded in Kurdistan
Region and most of them were found on gills of
cyprinid fishes (Mhaisen, 2014).
B
A
C
D
E
Fig. (1): Dactylogyrus skrjabensis
A- Photomicrograph of the worm
B- Photomicrograph of the copulatory organ
C- Photomicrograph of the haptor
D- Camera lucida drawing of the haptor
E- Camera lucida drawing of the copulatory organ
c= copulatory organ; co= connecting bar; e= eye spot; ha= haptor; meh=
median hook; mh= marginal hook; o= ovary; ph= pharynx; se= seminal
vesicle; sp= supplementary bar; t= testes; v= vetelaria
301
References
Ali, N. M., Al-Jafery A. R. and Abdul-Ameer K.
N. (1986). New records of three
monogenetic trematodes on some
freshwater fishes from Diyala river, Iraq,
J. Biol. Sci. Res., 17(2): 253-266.
Barzinji, K. T. M. (2013). Classification of
watersheds in Sulaimaniyah governorate
based on database of some morphometric
characteristics. IJPAES. 3 (2): 203-221
Bykhovskaya-Pavlovskaya, I. E., A. V.
Gusev, M.
N.
Dubinia,
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Izyumova, T. S. Smirnova, I. L.
Sokolovskaya, G. A. Shtein, S. S.
Shul'man, and V. M. Epshtein, (1962)
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U. S. S. R, Akad. Nauk, S. S. S. R.,
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Coad, B.W. (2010). Freshwater fishes of Iraq.
Pensoft Publ., Moscow: 274pp. + 16pls.
Gibson, D. I.; Timofaeva T. A. and Gerasev P. I.
(1996). A catalogue of the nominal
species of the monogenean genus
Dactylogyrus Diesing, 1850 and their host
genera. Syst. Parasitol., 35: 3-48.
Gussev, A.V.; Ali, N.M.; Abdul-Ameer, K.N.;
Amin, S.M. & Molnár, K. (1993). New
and known species of Dactylogyrus
Diesing,
1850
(Monogenea,
Dactylogyridae) from cyprinid fishes of
the river Tigris, Iraq. Syst. Parasitol., 25:
229-237.
Harris P. D. (1988). Changes in the site
specificity of G. turnbulli Harris, 1986
(Monogenea)
during
infection
of
individual guppies (Poecilia reticulate
Peters, 1859). Canadian J. Zool. ,
66:2854-2857.
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Hoffman, G. L. (1967). Parasites of North
American freshwater fishes. Univ. of
California Press, Bekeley.
Kritsky, D. C.; Panndey, K. C.; Agrawal, N. and
Abdullah, S. M. A. (2004). Monogenoids
from the gills of spiny eels (Teleostei:
Mastacembelidae) in India and Iraq,
proposal of Mastacembelocleidus gen. n.,
and status of the Indian species of
Actinocleidus,
Urocleidus
and
Haplocleidus
(Monogenoidea:
Dactylogyridae). Folia Parasitol., 51: 291298.
Margolis, L. and Kabata Z. (1984). Guide to the
parasites of fishes of Canada Part I.
National Printers Press,(Ottawa) Canada
Inc, 209 Pp.
Mhaisen, F. T. (2014). Index-catalogue of
parasites and disease agents of fishes of
Iraq. Unpuble. (Personal communication).
Neary, E. T., Develi N. and Özgül G. (2012).
Occurrence of Dactylogyrus species
(Platyhelminths,
Monogenean)
on
Cyprinids in Almus Dam Lake, Turkey.
Turkish Journal of Fisheries and Aquatic
Sciences, 12: 15-21 (2012)
Pugachev, O.N.; Gerasev, P.I.; Gussev,
A.V.; Ergens, R. and Khotenowsky, I.
(2010). Guide to monogenoidea of
freshwater fish of Palaeartic and Amur
regions. Ledizioni Ledipublishing,
Milano: 567pp.
Raissy, M. and Ansari, M. (2012). Parasites
of some freshwater fish from Armand
river, chaharmahal va Bakhtyari
province, Iran. Iranian J. Parasitol.,
7(1): 73-79.
@@ón‚íq
@Dactylogyrus skrjabinensis Osmanov, 1958@Žñí‚@óäb‚@Ûbm@õŠóÙäíØ@a†óîòìóåïÜüÙŽïÜ@ãóÜ
@õŠí’bi@óÜ@çaì@õŠbiììŠ@óÜ@óØ@ Capoeta trutta@ðbà@ñìí“îŠ@ŠóóÜ@a‹Ø@Šbàüm@×aÈ@óÜ@Šbu@ãóØóî@üi
@NRPQT@ ‰îìýóØ@bm@ ŒìŠìóä@çaíŽïä@ñòìbà@óÜ@Lòìómóäìa‹ØüØ@×aÈMçbnì†ŠíØ@ðáŽîŠóè@LðäbáŽïÝ@õŠb’@ôm
ýóèˆûŠ
@õ‡äím@ì@ çìíi@•ìím@õò‰ŽîŠ@óØ@o²Šò†@óØòìóåïÜüÙŽïÜ@Lòìímbè@a†óØòìóåïÜüÙŽïÜ@óÜ@óØòŠü‚ó“à@ñóäaíŽïq@ì@Òòì
@@@NÛóî@õaì†óÜ@Ûóî@٣٫٦٦@ì@٪٣٫٩٤@óÜ@çìíi@î‹i@çìíi@•ìím
@@
@@
@@
@óÔ‹¾a@óÝïnÜa@Ûba@â–þÌ@ôÝÈ@Dactylogyrus scrjabinensis@@d“å¾a@ñ†bya@ã‹ƒáÝÜ@ŠíéÄ@ßìa
@@×a‹ÉÜa@ÀCapoeta trutta
@@
@@ó–þ©a
@À@ ò‹à@ ßìý@ Dactylogyrus skrjabinensis Osmanov, 1958@ d“å¾a@ óî†byc@ ã‹ƒ¾a@ Þïvm@ @@@@@@
@æà@oÉ»@Üa@@Capoeta trutta (Heckel, 1843) óÔ‹¾a@óÝïnÜa@óÙ@â–þÌ@ôÝÈ@óïÜbä@óaŠ‡Üa@À@×a‹ÉÜa
@‹é’@¶a@Šaâ@‹é’@μi@òŠí—a@òÑÜa@ßþ‚@L×a‹ÉÜa@ Mçbn†ŠíØ@ âïÝÔa@LóïäbáïÝÜa@óÅÐb«@ðÔ‹’@líåu@Lçaì@‹éä
@ì@ ٪٣٫٩٤@ óib–fia@ ò‡’ì@ ójä@ oäbØì@ LóaŠ‡Üa@ ë‰è@ À@ ŠíØ‰¾a@ ðÝïÑÜa@ pbbïÔì@ Ò–ì@ ðÈc@ LRPQT@ la
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@
303
IDENTIFICATION OF POTATO VIRUS Y (PVY) AND ITS ECONOMIC
IMPORTANCE ON POTATO CROP
Nabeel Aziz Kassim 1, Zulaykha Abdulwahab Abduljalil Nerway 2 and Kurdistan Hassan Yousif 3
Department of Plant Protection, College of Agriculture and Forestry, University of Mosul, Mosul, Iraq
2
Department of Plant Protection, Faculty of Agriculture and Forestry, University of Duhok, Kurdistan Region, Iraq
3
Department of Horticulture, Faculty of Agriculture and Forestry, University of Duhok, Kurdistan Region, Iraq
(Accepted for publication: December 22 , 2014)
1
ABSTRACT:
This study was conducted to estimate disease incidence of potato virus Y (PVY) in Duhok Province/
Kurdistan Region/ Iraq and to investigate its effects on the growth and morphology of potato plant and its
productivity. High rates of occurrence of viral symptoms in the surveyed field were recorded. The mainly
included symptoms were mild to severe yellowing, mottling, necrosis, stunting and malformation of potato
plants. The effect of the virus on potato crop was studied using Vegetative growth and yield characters of
healthy, current season and tuber borne PVY infected plants. There is differentiation between the growth
of the current season, tuber borne PVY-infected and the virus free potato plant. Results showed that
infection by PVY leads to reduce many physiological functions of above and underground parts of host
plant like size of leaf area, chlorophyll percentage, number of tubers, tuber weight and total yield of a
plant. Depending on the results, because of reducing physiological functions of above ground part of
potato plant (leaf area and chlorophyll percentage), the number and the weight of tuber decreased, so the
productivity of the plant decreased.
KEYWORDS: Date of Infection, Potato, PVY, Vegetative growth and Yield Characters.
INTRODUCTION
Potato (Solanum tuberosum L.) is one of the
important world food and vegetable crops
belongs to the Solanaceae family (United
Nations Food and Agricultural Organisation,
2009) which planted commercially in Iraq since
1960 (Mattlob et al., 1989). In this respect, it
ranks the fourth world crop with a rate of nearly
325 million tons annual production (Nagib et al.,
2003).
Potato is a rich crop of nutrient substances so
it is consumed in very large quantities. Each 100
g of potato tuber contains 72-75 g water, 2- 2.5 g
protein, 0.15 g fatty acids, 16-20 g starch and 11.8 g fibers as well as it contains a little quantity
of nutrient elements and some vitamins. It
contains 0.17 mg thiamin, 0.4 mg Riboflavin,
2.2 mg Niasin and 42 mg vitamin C (United
Nations Food and Agricultural Organisation,
2009).
Potato tubers can transfer many diseases and
pests and these cause degeneration of the seed
tuber and plants. Potato production is being
seriously hampered due to certain viruses (Rolot
and Seutin, 1999), like potato virus Y (PVY)
which is the most dangerous virus. This virus
was detected in commercial fields in single or
mixed infection (Nascimento et al., 2003 and
Biswas et al., 2005).
PVY belongs to Potyvirus genus from
Potyviridae family (Posada and Crandall, 2001).
Its symptoms on potato ranged between mosaic
to necrosis and death of plants depending on
cultivar and viral strain (Robert et al., 2000).
PVY is widespread in Iraq on potatoes and other
plants (Al-Sameae, 2000; Kassim and
Mohammad, 2002 and Kassim and Younis,
2003). This study aims to survey PVY in Duhok
province/ Kurdistan region/ Iraq and to know the
effect of the virus on potato crop on the basis of
infection date.
1. Field Surveying and Sampling of PVY
Isolates
Ten donums of potato yield in Gre-gawre
village/Duhok province planted with Santa
cultivar were surveyed from April-July 2013,
using X pattern. Surveying were done every ten
days depending on visual observation of virus
symptoms. Leaf samples were collected and kept
in a deep freezer (-18 ºC) for detecting the virus
using double antibody sandwich enzyme linked
immunosorbent assay (DAS-ELISA) according
to that of Koenig et al. (2008).
304
2. Plant Material
Three groups of potato plants selected in the
field due to PVY in the following orders as well
as tuber borne PVY, current season PVY and
PVY-free. The plants in the first group were
carried tuber borne PVY while the second group
include the plants in fact were healthy but
infected by PVY because of feeding of green
peach aphid (Myzus persicae). PVY-free group
includes healthy plants. To ensure the presence
of the virus, all used plants were tested using
double antibody sandwich enzyme linked
immunosorbent assay (DAS-ELISA) (Koenig et
al., 2008).
3. Experimental Measurements
At the mid and end of season of growth of
2014, several characteristics of the plants were
taken to determine the effect of PVY on plants
depending on date of infection and compare
them with control (healthy) potato plants.
3.1. VEGETATIVE GROWTH
CHARACTERS
-1
3.1.2. Percentage of Chlorophyll Content of
Leaves
It was determined after 55 days from planting
from several plants of inner rows in each group
by using Chlorophyll Meter (Adrijana et al.,
2008).
3.2. YIELD CHARACTERS
3.2.1. Number of Tubers.Plant-1
The number of tubers per plant was counted
from each group at the end of the growing
season.
3.2.2. Tuber Weight (kg.Tuber-1) and Total
Yield (kg.Plant-1)
The average weight of tuber was obtained by
weighting the tuber of each group at harvest then
divided by the tuber number in each
experimental unit.
RESULTS AND DISCUSSIONS
2
3.1.1. Leaves Area.Plant (cm )
The leaf area per plant was measured. It was
measured before harvesting in a randomly taken
samples represented by several physiologically
completed leaves of several plants from each
group. Three discs were taken from each leaf
and the average of each disc was counted and
weighted. Moreover, the fresh weight of the disc
and the leaf was taken. Then, on the basis of
proportion ratio the leaf area was counted and
the average of the leaf area was calculated (Bn
Sultan, 1996). Single leaf area =Weight of the
305
leaf area (g) × Known area of the leaf section
(1cm2) / Weight of the sections (g).
1. Field Surveying and Sampling of PVY
Isolates
The definitive symptoms observed in the
surveyed field were severe mosaic, necrosis,
yellowing and mottling. Disease incidence of
such field was determined in the figure. The
results showed that the disease incidence was
high in potato crop in the beginning of the
season and gradually increased to the end of
season.
Figure (1): Disease Incidence of PVY in Surveyed Field
Virus disease incidence in potato field
increased because of two reasons. Planting of
uncertified potato seeds and the use of the tubers
produced in the previous seasons which were
heavily infected by the virus cause to a serious
degradation of potato plants grown from such
tubers and cause to increase virus disease
incidence. This was in agreement with that of
Jones et al. (2003) and Chatzivassiliou et al.
(2008) who found that PVY was the more
commonly spread virus through tubers harvested
from infected potato plants. Hamm and Hane
(1999) stated that disease incidence was
increased by using viral infected potato seeds.
Another reason that caused gradually increase
in the disease incidence from the beginning of
the season to the end is green peach aphid
(Myzus persica) which transmit PVY from
diseased plant to healthy one. Study of Boiteau
et al. (1998) was in agreement with our result.
Myzus persica has been found to be most
effective aphid in its role as a vector for PVY
(Warren et al., 2005). Sławomir (2010) stated
that PVY is active after 17 hours of its
acquisition on the aphid's stylet, so the
epidemiology of the virus was increased. On the
other hand, planting of the potato tubers that
carry the virus and the presence of green peach
aphid (Myzus persica) in potato field lead to
increase the incidence of the viral disease of
potato plants.
2. EXPERIMENTAL MEASUREMENTS
2.1. Vegetative Growth Characters
2.1.1. Leaves Area.Plant-1 (cm2)
The average leaf area of healthy, tuber borne
and current season PVY infected plants shows in
table (1). Results showed that leaves area of
infected plants significantly are smaller than
control plants (6.42 cm2). Leaf area of current
season and tuber borne PVY infected plants
were 4.30 and 2.33 cm2, respectively.
Table (1): Effect of Current Season and Tuber Borne PVY Infection on Leaf Area (cm2) and Total Chlorophyll
Content (%) of Potato Plants Compared to Healthy Plants in Control
Trails
Leaf Area (cm2)
Control
(PVY-Free Plants)
6.42 a
Treatment
Current Season
PVY Infected Plants
4.30 b
Tuber Borne PVY
Infected Plants
2.33 c
Total Cholorophyll Content %
43.55 a
33.46 b
28.21 c
306
The average leaf area of current season and
tuber borne PVY infected plants showed that the
virus has a great effect on leaf area of infected
plants compared to control plants. As noticed in
the result, the effect of the virus is higher on the
tuber borne infected plants than current season
infected one and there are significant differences
between leaf area of different types of plants.
The result was in agreement with Fargette et al.
(1988) and Hooks et al. (2008). They stated that,
there is a significant difference in the size of leaf
area between healthy and current season and
tuber borne viral infected plants. They showed
that certain aspects of plant growth may be
affected by virus infection.
2.1.2. Total Chlorophyll Content of Leaves
(%)
The average of total chlorophyll content of
the virus free, tuber borne and current season
PVY infected plants shows in table (1). It is
noticed that total chlorophyll content of current
season, tuber borne PVY infected and control
plants were 33.46%, 28.21% and 43.55%,
respectively. In the results, the significant
differences between total chlorophyll content of
healthy (control), current season and tuber borne
PVY infected potato plants can be noticed. Our
result is in agreement with Hooks et al. (2008)
and Jakab-Ilyefalvi (2008). They found
significant differences in total chlorophyll
content of these different types of potato plants.
2.2. YIELD CHARACTERS
2.2.1. Number of Tubers.Plant-1
The average number of tubers.plant-1 of
different types of potato plants shows in the
following table (2). Results showed that the
number of tubers for each of PVY-free plants
was as much as 9.60 compared to other types of
plants (current season and tuber borne PVY
infected plants) as well as 8.20 and 3.60,
respectively.
Table (2): Effect of Current Season and Tuber Borne PVY Infection on Number of Tubers, Tuber Weight (kg)
and Total Yield (kg) of Potato Plants Compared to Healthy Plants in Control
Trails
No. of Tuber.Plant-1
Tuber Weight (kg.tuber-1)
Total Yield (kg.plant-1)
Control
(PVY-Free Plants)
9.60 a
0.40 a
3.80 a
2.2.2. Tuber Weight (kg.Tuber-1) and Total
Yield (kg.Plant-1)
The average weights of a tuber and the total
yield of a healthy, tuber borne and current
season viral infected plant showed in the
previous table (Table 2). As appeared in the
table, tuber borne PVY infected plants had the
lower average weight of a tuber and total
yield.plant-1 (0.10 and 0.44 kg) compared to
current season infected (0.12 and 0.80 kg) and
control plants (0.40 and 3.80 kg). There are
significant differences between different types of
plants. As noticed to the results showed in the
table (2), any increasing or decreasing in the
number and the weight of a tuber.plant-1 leads to
increase or decrease the average productivity of
the plant, respectively as well as 3.80, 0.80 and
0.44 kg tubers as the final productivity of
healthy, current season and tuber borne PVY
infected plants. The result was in agreement with
that of Fargette et al. (1988). Host plants have a
wide range of responses to PVY infection. In
307
Treatment
Current Season
PVY Infected Plants
8.20 a
0.12 b
0.80 b
Tuber Borne PVY
Infected Plants
3.60 b
0.10 b
0.44 c
fact, these responses were determined by potato
cultivar and virus strain, and whether there is
primary or secondary infection (Nie et al.,
2012). Virus infection has negative effects on
plants by limiting their growth (Miteva et al.,
2005). Yield reduction of an infected plant with
a virus was greater when plants were infected
from the vegetative propagation materials than
later by the vector (Fargette et al., 1988). In the
present study, there were several measurements
of potato growth significantly differ from tuber
borne and current season PVY-infected plants to
control one. Fargette et al. (1988) showed that
some properties of plant growth may be affected
by virus infection. The symptoms caused by
virus like mosaic surfaces, necrotic zones and
reducing the size of leaf area lead to reduce
chlorophyll content (Jakab-Ilyefalvi, 2008).
Alterations in the biosynthesis of chlorophylls
cause low chlorophyll content of infected plants.
This has negative effect on the physiological
factors including the metabolic processes.
Physiological disorders associated with low rate
of photosynthesis lead to decrease the total
chlorophyll content of infected vegetative parts
of plants and this cause to reduce the
productivity of infected plants (Chia & He, 1999
and Hook et al., 2008).
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309
IN VITRO MULTIPLICATION OF PHOTINIA (PHOTINIA X
FRASERI)USINGDIFFERENT CULTURE MEDIA
Layla Shaaban Mohammed AL-Mizory and Ameena Mohammed Hassan
Horticulture Dept., Faculty of Agriculture and Forestry, Duhok University, Kurdistan Region, Iraq.
ABSTRACT:
A very high rate of multiple shoots was obtained from nodal explants of Photinia x fraseri on MS
medium supplemented with a various concentration of Kin alone (1, 2, and 3) mgl-1alone and combination
of Kin at (1, 2, and 3) mgl-1 with IBA or IAA at (0.2 and 0.4) mgl-1. The results show that the number of
shoots/ explant was significantly affected the levels of Kin alone and combination with IBA or IAA. The
number of shoots (6.00 shoot/ explant) when Kin added to the medium at 3mgl-1 in which it was
significantly different from other Kin concentrations. While the effect of different medium with the fixed
and best concentration of Kin+ IBA and Kin+ IAA on the average shoot number and leaves number
/explant of Photinia explant (node segment and shoot tips) The highest number of shoots (5.90) was
recorded in node segment when cultured on WPM containing Kin+ IBA and it was increased significantly
compared with all treatments from shoot tip. However, the maximum number of leaves (20.10) was
recorded on WPM medium supplemented with Kin+ IBA and it was increased significantly compared
with all treatments which containing different media plus Kin at 2mgl-1 + 0.4mgl-1 IBA and Kin at 1mgl-1+
0.4mgl-1 IAA.Shootlel form the combination of from photinia cultured on ¼MS medium improved 0.6 mgl1
NAA concentration produced the highest number of roots/shootlel (6.5roots/shootlet) compared with all
treatments contains ½MS medium added IAA, IBA and NAA on all concentration. While, theshootlets
from photinia cultured on ¼MS medium and supplemented with 0.6mgl-1NAA formed an average (5.29
cm) compared to all treatments containing ½MS medium. Such plantlets were successfully transferred to
soil after hardening with a high rate of survival.
Keyword: Photinia x fraseri, tissue culture media and plant growth regulators.
INTRODUCTION:
F
raser Photinia belongs to Rosacea family
it is a general evergreen shrubs with
glossy green leaves, white flowers and young
red shoots. Photinia is a genus of about 40-60
species of small trees and large shrub. They are
restricted to warm temperature in Asia, from the
Himalaya east to Japan and to India and
Thailand (James, 1992). Fraser Photinia is an
important woody landscape plant used for
hedging and screening in the USA (Dirr, 1983
and Dirr and Heuesr, 1987). Red tip is used to
create majestic tall hedges. Red tip hedges can
be left unprimed for “natural” look. They retain
foliage to the ground and never become leggy.
The traditional propagation method of
photinia is by rooting the apical cutting with
great concentrations of plant growth regulators
(Beeson, 2000). The elongated period required
to obtain new plant and the rooting difficulty of
cutting are some of the factors that limited the
commercial exploitation of this species
(Larraburuet al., 2007). Red tip of photinia can
be propagated using seed exposed to a twomonth cold stratification; another method for
photinia propagation is by tissue culture
technique but the current information on the
application of in vitro technique for the
multiplication of this species is very limited
(Kaneet al., 1987; Leifertet al., 1992).The
production of woody plants by tissue culture
methods is used to facilitate rooting and used to
propagation species with rooting difficulties, to
solve problems of the seasonal supply associated
with the rooting of stem cuttings and to clone
disease-resistant specimens and to provide target
material for gene transfer (Merkle and Dean,
2000). Terminal and lateral shoots of photinia
were cut in to node segment of similar size
(1cm) and transferred to 300ml flasks with 70ml
of medium, supplemented with different
concentration of cytokinin according to Leifertet
al. (1992) and Rafael et al. (1997).The shoot
length of photinia increased significantly when
cultured on MS medium containing 2mgl-1 BA
with average of 23 mm at 28 day of growth and
the highest multiplication rate (4.30) shoots were
achieved (Larraburnet al., 2007). The medium
for multiplication of Ficushietawas ½MS +
0.5mgl-1BA (Jiang et al., 2004). The highest
number of shoots (5 shoot/ explants) of
concentration was formed on MS medium
containing 4mgl-1BAP compared to (0.69shoots)
310
on basal medium. However, shoot regeneration
indifferent concentration of Kin was nearly
constant (1.18 shoot/ explant). In most
concentration of cytikinine, Kin led to produce
longer shoots in comparison with BA and as the
concentrations of cytokinine increased, the
height of shoots decreased (Kharrazi et
al.,2011).
Behera and Sahoo (2009) found that the best
performance was achieved on MS medium
supplemented 2mgl-1BA + 0.5mgl-1IBA on this
combination, 92% of the explants produced
shoots. Choudharyet al. (2011) found that when
increased the concentration of BAP in the
nutrient media for Aloe vera (1BAP and
0.5NAA) mgl-1, the number of shoots were also
increased per culture. Photinia were rooted on
half strength WPM containing 0.02mgl-1 NAA
(Lloyd and McCown, 198٠). Larrabur net
al.(2010) used MS medium for photinia shoots
rooting supplemented with 10 mg-1IBA.While
the best result if root formation was observed on
MS medium containing 5mgl-1 NAA (Tariqueet
al. 2010). Das (2010) indicated that the
maximum percentage of rooting for rose microshoots (94%) was noted on medium having half
strength of MS medium with 0.25mgl-1 IBA
within 15 days of cultures.
The aim of study is to identify a suitable
explant for shoot induction, a suitable culture
media, suitable plant growth regulators and their
concentration of shoot multiplication and
rooting.
MATERIAL AND METHODS:
The current study was carried out in the
Laboratory of plant tissue culture, faculty of
Agriculture, university of Dohuk, Kurdistan
Region, Iraq, during the period from December
2013 to May 2014.Node segment and shoot tip
was used as an explant in this investigation
which was taken from an in vitrosterile explant
and then conducted the following experiments:
For multiplication stage.
1. MS + Kin (1, 2, and 3) mgl-1alone.
2. MS + Kin (1, 2, and 3) mgl-1 combination
with IBA or IAA at (0.2 and 0.4) mgl-1.
3. The best concentrations from Kin
combination with IBA or IAA add to the
different media like (WPM, B5, White and
DKW).
All culture media (MS, WPM, B5, White and
DKW)MS (Murashige & Skoog 1962),WPM
311
(Lloyd and McCown 1980), B5 (Gam-bor get al.
1968), DKW (Driver and Ku-niyuki 1984),
supplemented with 3% sucrose,0.7% agar. After
6-8 weeks, the following data were recorded:
Number of shoots, leaves, nodes and Shoot
length.
For rooting stage conducted the following
experiments:
1. (½, ¼) MS with (0.0, 0.6 and 0.8) mgl1
IBA or IAA or NAA alone.
After 8 weeks, the following data were
recorded: Root percentage, Number of root and
Root length
Acclimatization stage: After 8 weeks of shoot
rooting the plantlets were thoroughly washed
with tap water to remove the agar from roots
which might be a source of concentration. The
plantlets were put in pots containing autoclaved
mixture of peat moss and sand in ratio of (1:1/ v:
v) pleased in sterile boxes covered by
polyethylene in order to maintain high relative
humidity. The potted plants were placed in
incubation room for 30 days. After 4weeks the
plants transferred to the green house.
Data Analysis:
The experiments were arranged according to
Complete Randomized Design (CRD) using (5)
replication for each treatment. Data were
analyzed and means were compared with each
other using Duncan’s multiple rang test at 0.05
level (Duncan, 1955).
RESULT AND DISCUSSION
Effect of Kin concentration alone and
combinations with IBA or IAA on
multiplication stage of Photinia explants:
To
determine
the
most
suitable
concentrations of Kin, IBA or IAA and their
combinations on shoot multiplication, the
explant were excised and inoculated on MS
medium containing different concentrations of
Kin with IBA or IAA. As it is clarified in Table
(1) the effected of various concentrations of Kin,
IBA or IAA and their combinations on shoot
number, leaf number, number of node and shoot
length of photinia explant after 8 weeks of
culture to multiplication stage. It reveals
different concentration of Kin, IBA or IAA and
their combinations on shoot number. The results
show that number of shoots/ explant was
significantly affected at levels of Kin alone and
combination with IBA or IAA. The number of
shoots (6.00 shoot/ explant) was observed when
Kin added to the medium at 3mgl-1which is
significantly different from other Kin
concentrations.
The combinations between Kin and IBA or
IAA increased significantly shoot number to
(5.700 and 4.200) shoot/explant when 2mgl-1
Kin + 0.4mgl-1IBA and 1mgl-1Kin +0.4mgl-1IAA
respectively, when added to the MS medium
compared with all treatment except the treatment
1mgl-1Kin + 0.4mgl-1IBA.Whereas, the less
number of shoots (1.500) was found on MS
medium free hormones. As it is illustrates from
the same table (1) that the effect of different
concentration of Kin separately, IBA and IAA
together on number of leaves, number of node
and shoot length. The maximum number of
leaves and number of nodes (20.100 leaves/
explant and 5.100 node/ explant) were obtained
on MS medium supplemented with 3 mgl-1Kin.
While the highest length of shoots (4.41cm) was
found when 2mgl-1Kin added to the MS medium.
However, the minimum number of leaves,
number of nodes and shoot length (10.500
leaves/ explant 2.200node/explant and 1.55cm)
respectively, were found on MS medium free
hormones. About the combinations between Kin
concentrations with IBA or IAA produced of
number of leaves; number of nodes and shoot
length were increased on the combinations. The
maximum number of leaves (19.80 and 17.30)
leaves /explant when (2mgl-1Kin +0.4mgl-1IBA)
and (1mgl-1Kin+0.4mgl-1IAA) were added to the
MS medium, respectively. While the minimum
number of leaves (13.900 and 13.800 leaves/
explant) was found on MS medium
supplemented with 2mgl-1Kin+0.2mgl-1IBA and
3mgl-1Kin +0.4mgl-1IBA respectively. On the
other hand, the interaction also revealed
significant difference and the treatment of Kin at
2mgl-1+ IBA at 0.4mgl-1gave the highest number
of nodes (5.100 node/explant). However, the
interaction between Kin concentrations with IBA
or IAA concentrations gave the maximum
number of nodes (4.00 node/explant) was
obtained when Kin at 1mgl-1+ 0.4 mgl-1IAA was
added to the MS medium. Whereas, the less
number of node (2.80 node/ explant) was
observed on MS medium containing 1 mgl1
Kin+0.2 mgl-1IBA. The results clarify that the
shoot length was significantly affected when
using the interaction between Kin with IBA or
IAA. While, using MS medium containing 3mgl1
Kin+0.4mgl-1IBA and 3mgl-1Kin+0.2mgl-1IAA
which gave highest length of shoot (3.85 and
3.91) cm was more affected increased
significantly the shoot length when compared
with majority and control treatments which gave
the lower shoot length. Treatment which gave
minimum length of the shoots length was
observed on MS medium plus 1mgl-1`Kin +
0.2mgl-1IBA which amounted to (2.030 cm). The
figure (1) illustrates the effect of different
concentrations of Kin, IBA and IAA at
multiplication stage after 8 weeks of culture.
Similar response was observed with
Tiliaplatyphyllosscop (Üçler et. al.2001). The
minimum number were formed with MS
medium free growth regulators (control) and MS
medium having 3 mg L-1 Kin in tissue cultures
(in addition to in integral plants and plant
organs), cytokininsseem to be necessary for
plant cell separation. Cytokinins are very active
in encouraging direct or indirect shoot
development.
312
Table (1): Effect of Kin alone, IBA and IAA combination with Kin on multiplication of photinia explant culture
on MS medium after 8 weeks.
PGRs
PGRs
Conce. Mgl-
Shoot Number
Leaves
Number
Node Number
Shoot Length
(cm)
control
Kin
Kin
Kin
Kin+IBA
Kin+IBA
Kin+IBA
Kin+IBA
Kin+IBA
Kin+IBA
Kin+IAA
Kin+IAA
Kin+IAA
Kin+IAA
Kin+IAA
Kin+IAA
1
2
3
1+0.2
2+0.2
3+0.2
1+0.4
2+0.4
3+0.4
1+0.2
2+0.2
3+0.2
1+0.4
2+0.4
3+0.4
1.500e
2.600de
4.100bc
6.00a
3.100cd
3.700cd
4.200bc
5.200ab
5.700a
4.200bc
3.300cd
3.400cd
3.100cd
4.200bc
4.100bc
3.500cd
10.500d
13.600cd
15.400abcd
20.100a
14.100cd
13.900cd
15.700abc
17.600abc
19.800ab
13.800cd
14.500cd
16.100abc
14.300cd
17.300abc
15.100abcd
14.900bcd
2.200e
3.100cde
3.000cde
5.100a
2.800de
3.600bcd
4.100abcd
4.600ab
5.100a
4.300abc
4.000abcd
3.200bcde
3.900abcd
4.000abcd
3.700abcd
3.200bcde
1.550d
3.480ab
4.410a
3.120bc
2.030cd
2.240cd
3.620ab
3.590ab
3.700ab
3.850ab
2.870bc
2.030cd
3.910ab
3.670ab
2.820bc
2.180cd
1
* Means followed by the same letter within each character (column) do not differ significantly (P≤0.05)
according to Duncan's Multiple Range Test (Duncan, 1955)
Effect of different medium with the fixed and
best concentration of Kin+IBA and Kin+
IAAon the average shoot number and leaves
number /explant of Photinia explant (node
segment and shoot tips).
Table (2) showed the effect of different type
of media supplemented with fixed concentration
of (Kin at 2mgl-1 +0.4mgl-1 IBA and Kin at
1mgl-1+0.4mgl-1 IAA) on shoot number and
leaves number from two types of explant (node
segment and shoot tips).
Regarding means value of explants show, the
maximum number of shoots (4.39 shoots/
explant) from node segment was significant
compared with shoot tips (3.88 shoot/ explant).
While the means value of type of media plus
PGRs at fixed concentration, the maximum
313
number of shoots (5.75) shoot/ explant was
significantly increased when the explant cultured
on WPM medium plus Kin +IBA compared with
all treatments. While the highest number of
shoot (4.60) was observed on WPM medium
containing Kin+IAA but the increase was not
significantly. Whereas, the minimum number of
shoots (3.15) was found on White medium
supplemented with Kin at 1 mgl-1+IAA at 0.4
mgl-1respectively.
The interaction between the explant and types
of medium containing fixed concentration of
Kin+ IBA and Kin+ IAA was significantly
increased shoots number. The highest number of
shoots (5.90) was recorded in node segment
when cultured on WPM containing Kin+ IBA
and it was increased significantly compared with
all treatments from shoot tipin control treatment
when shoot tip cultured on WPM containing
Kin+ IBA. In addition the maximum number of
shoot (5.60) was observed for the shoot tip
cultured on the same medium. While, the lest
number of shoot (3.00) was obtained from shoot
tip when cultured on White medium containing
Kin+ IAA.
On the other hand, the mean values of
experimental results regarding on the same table,
the highest number of leaves from node segment
and shoot tip were (15.6 and 13.98) respectively,
the node segment was increased significantly
compared with shoot tip. The maximum number
of leaves (20.10) was recorded on WPM
medium supplemented with Kin+ IBA and it was
increased significantly compared with all
treatments which containing different media plus
Kin +IBA and Kin+IAA. Whereas, the minimum
number of leaves (13.10) were found on B5 and
DKW media containing Kin +IAA.
The combinations between the explant and
types medium containing fixed concentration of
Kin+ IBA and Kin+ IAA to leaves emergence
show that the maximum number of leaves
emergence from node segment (20.60) and from
shoot tip (19.60) was found on WPM medium
supplemented with Kin+ IBA and this treatments
was significantly increased with all treatment for
shoot tip and some treatments for node segments
when culture on WPM, B5, DKW and White
media containing Kin+ IBA. However, the less
number of leaves (12.70) was obtained from
shoot tips when cultured on white medium
containing Kin+ IBA.The figure (2) illustrates
the effect of different type of media with fixed
concentrations of Kin, IBA and IAA on node
segment and shoot tips at multiplication stage
after 8 weeks of culture.
Table (2): Effect of different type of media with fixed concentration of Kin+ IBA and Kin+ IAA on the average
shoot number and leaves number /explant of Photinia explant (node segment and shoot tips) on multiplication
stage after 8 weeks.
PGRs
Media
WPM
B5
Kin+IBA
DKW
White
WPM
B5
Kin+IAA
DKW
Shoot Number
Node
Shoot
seg.
tip
5.90a
5.60ab
4.80abc
4.00cde
d
4.20bcd
3.70de
e
3.50de
3.40de
5.30abc 3.90cde
3.80de
3.10e
4.30abc 4.30abc
d
d
3.30de
3.00e
MeansType of
Media with
PGRs
5.75a
Leaves Number
Node
Shoot
seg.
tip
20.60a
19.60a
Means Type of
Media with
PGRs
20.10a
4.40bc
16.10ab
13.50b
14.80b
3.95bcd
16.20ab
13.10b
14.65b
3.45cd
4.60b
3.45cd
14.40ab
17.90ab
13.40b
12.70b
13.30b
12.80b
13.55b
15.60b
13.10b
4.30bc
12.80b
13.40b
13.10b
White
3.15d
13.40b
13.40b
13.40b
Means of
4.39a
3.88b
15.60a
13.98b
Explants
314
Effect of different medium with the fixed and
best concentration of Kin+ IBA and Kin+ IAA
the average node number /explant and shoot
length (cm) of Photinia explant (node segment
and shoot tips).
It is shown from Table (3) the effect of
different type of media supplemented with fixed
concentration of (Kin at 2mgl-1 + 0.4mgl-1 IBA
and Kin at 1mgl-1+ 0.4mgl-1 IAA) on number of
node and shoot length form two types of explant
(node segment and shoot tips).
Regarding means value of explants show; the
maximum number of node (4.40node/ explant)
from node segment was significant compared
with shoot tips (3.79node/ explant). While the
means value of type of media plus PGRs at fixed
concentration, the maximum number of
nodes(5.70) shoot/ explant were significantly
increased when the explant cultured on WPM
medium plus Kin +IBA compared with all
treatments. While the highest number of node
(5.00) was observed on WPM medium
containing Kin+ IAA but the increased was not
significantly. Whereas the minimum number of
node (3.15) was found on White medium
supplemented with Kin +IBA and Kin +IAA
respectively.
The interaction between the explant and types
of medium containing fixed concentration of
Kin+ IBA and Kin+ IAA was significantly
increased. The highest number of shoots (6.40)
was recorded in node segment when cultured on
WPM containing Kin+ IBA and it was increased
significantly compared with all treatments from
shoot tip. In addition the maximum number of
node (5.00) was observed from shoot tip when
the shoot tip cultured on the same medium.
While, the lest number of node (3.00) was
315
obtained from shoot tip when cultured on White
medium containing Kin+ IAA.
On the other hand, the mean values of
experimental results regarding on the same table,
the highest length of shoots from node segment
and shoot tip (3.42 and 4.09) cm respectively,
the shoot tip was increased significantly
compared with node segment. While the mean
values of different type media containing fixed
concentration of Kin+ IBA and Kin+ IAA. The
maximum length of shoots (4.79cm) was
recorded on WPM medium supplemented with
Kin+ IBA and it was increased significantly
compared with all treatments which containing
different media plus Kin +IBA and Kin + IAA.
Whereas, the minimum length of shoots
(3.05cm) were found on white media containing
Kin+ IAA.
The combinations between the explant and
types medium containing fixed concentration of
Kin+ IBA and Kin+ IAA length emergence
shows that the highest length of shoots
emergence from shoot tip (5.28cm) and from
node segment (4.31cm) was obtained on WPM
medium improved with Kin+ IBA and this
treatments was significantly increased with all
treatment for node segment and some treatments
for shoot tip when culture on WPM, B5, DKW
and White media containing Kin+ IBA.
However, the less number of leaves
(3.05cm)were obtained from node segment and
shoot tips when cultured on while medium plus
Kin+ IBA and Kin +IAA. The figure (3)
illustrates the effect of different type of media
with fixed concentrations of Kin, IBA and IAA
on node segment and shoots tips at
multiplication stage after 8 weeks of culture.
Table (3): Effect of different type of media with fixed concentration of Kin+ IBA and Kin+ IAA on the average
node number /explant and shoot length (cm) of Photinia explant (node segment and shoot tips) on multiplication
stage after 8 weeks.
PGRs
Media
WPM
B5
Kin+IBA
DKW
Node Number
Node
Shoot
seg.
tip
6.40a
5.00bc
4.50bcd
4.80bcd
e
4.50bcd
3.30de
e
3.20e
3.10e
5.80ab
4.20cde
3.90cde 3.60cde
3.60cde
3.30de
3.00e
3.30de
Means Type of
Media with
PGRs
5.70a
Shoot Length (cm)
Node
Shoot
seg.
tip
4.31abc
5.28a
Means Type of
Media with
PGRs
4.79a
4.65bc
3.21bc
4.54ab
3.88bc
3.90cd
3.32bc
4.33abc
3.83bc
White
3.15d
3.05c
4.51ab
3.78bc
WPM
5.00ab
3.97abc 4.41abc
4.19ab
Kin+IAA
B5
3.75cd
3.21bc
3.43bc
3.32bc
DKW
3.45d
3.24bc
3.24bc
3.24c
White
3.15d
3.05c
3.05c
3.05c
Means of
4.40a
3.79b
3.42b
4.09a
Explants
Effects of salt strength of MS media and
different concentrations of IAA, IBA and NAA
on rooting stage
1. Rooting percentage
The percentage of root was significantly
affected by the various treatments tested on
photinia (Table 4)The mean value of type of
auxin show that the highest percentage of root
formation when NAA used and gave the better
results on root percentage formation (98.33%)
than IBA and IAA. Whilethe mean value of type
media, the percentage of root on (84.29%) was
detected as a result of amending the ¼MS
medium compared with ½MS medium (81.5%)
and it was significantly increased. However, the
mean values of auxin concentrations show that
the highest percentage of root development
(100%) was observed when 0.8mgl add to the
medium. A similar results was reported by
(Danial et. al. 2008)by using of MS medium
with(0.5mgl-1)NAA encouraged root growth
(5.67 ± 1.15) and MS medium containing 1mgl1
IBA gave (2.15 ± 1.02)of Dianthus
Caryophyllus L culture.
The effect of the three interactions (diverse
media concentrations, various kinds of auxine
and their concentrations) showed that rooting
percentage could be achieved with the use of
basic ¼MS medium and ½MS medium
increased with NAA, IBA and IAA
concentrations. The explants of photinia cultured
on ¼MSmedium supplemented with (0.2, 0.6
and 0.8 )mgl-1NAA and (0.6 and 0.8 ) mgl-1IBA
concentration produced an average of (100%)
rooting compared with ½MS medium
316
supplemented with all concentration of NAA,
IAA and IBA concentration. Oppositely, the
lowest percentage of roots were made on
explants (42.5%) with ¼MS and ½MS medium
without hormones.
Table (4): Effect of salt strength of MS media and different types of auxin concentrations on roots response of
photinia after 8 weeks.
Conce.
Mgl-1
Control
0
IAA
0.2
IAA
0.6
IAA
0.8
IBA
0.2
IBA
0.6
IBA
0.8
NAA
0.2
NAA
0.6
NAA
0.8
Means of Media
Auxins Type
% Root
½MS
¼MS
42.5f
42.5f
77.5de
85abcde
75e
87.5abcde
80cde
82.5bcde
80cde
95abc
80cde
100a
90abcde
100a
92.5abcd
100a
97.5ab
100a
100a
100a
81.5b
84.29a
Means of
Auxinsconce
81.25c
81.25c
81.25c
87.5bc
90abc
95ab
96.25ab
98.75a
100a
Means of Auxins
Type
81.25a
90.83a
98.33a
2. Roots number
As presented in Table (5) Fig (4,5), data
shown that there was a significant variance in
the number of roots formed on shootlets of
photinia as a consequence of tested treatments.
The mean value of type of media show that the
highest number of roots/shootlet (4.76) was
recorded with ¼MS medium compared with
½MS medium (4.53) roots / shootlet and it was
significant increasing auxin concentrations.
However, the mean value of type of auxin show
that the highest percentage of root formation
when NAA used and gave the better results on
root percentage formation (5.66) and
significantly increased the number of root as is
compared to IBA and IAA . Data concerning the
mean result of auxin concentration treatments
and it’sthe maximum number of roots/shootlel
(6.05) was recorded for shootlelwith 0.6 mgl1
NAA, while, the lowest value (2.50) was
recorded 0.6 mgl-1IAA.
Consequences regarding the effect of the
three factors (diverse salt of MS medium
concentrations, kinds of auxin and auxin
concentrations) showed that the roots /shootlet
could be found with the use of unchanged ½MS
medium and ¼MS medium complemented with
NAA, IAA and IBA concentrations. Data also
317
indicated that IBA and NAA concentration when
extra to the ¼MS has the pronounced and
significant consequence on this parameter.
Shootlel from photinia cultured on ¼MS
medium improved with 0.6 mgl-1NAA
concentration formed the maximum number of
roots/shootlel (6.5roots/shootlet) compared with
all
treatments
having
½MS
medium
complemented with IAA, IBA and NAA on all
concentration. Otherwise, the lowest number of
roots on photinia explants (2.3 roots/ shootlet)
and was recorded with ½ MS medium having
0.2mgl-1IAA. Consequences under discussion
are in harmony with those described by (Ali et.
al. 2008). They explained that in order to
develop in vitro adventitious rooting, the isolated
plantlets were cultured on media having 0.1, 1.0
and 10.0 mg/l IAA or NAA in several physical
conditions. Optimum adventitious rooting and
succeeding plant survival was found by culturing
plantlets in medium having 0.1 mg/l NAA for 816 weeks prior to transplanting to soil (Tisserat,
1982). Date palm plants may be obtained by
transferring separate plantlet to MS medium
supplemented with 0.1 mg/l NAA to improve
rooting and 0.01mg/l BA to improve shoot
structure(Omar.1988).
Table (5): Effect of salt strength of MS media and different types of auxin concentrations on average root
number formed on shootlet of photinia after 8 weeks.
Auxins Type
Conce. Mgl-
Control
0
IAA
0.2
2.3f
IAA
0.6
3.6def
IAA
IBA
0.8
0.2
4.2cd
4.7bcd
4.4cd
4.7bcd
4.3cd
4.7bc
IBA
0.6
4.9bcd
5.4abc
5.15abc
1
Number of Root
½MS
¼MS
2.6ef
2.6ef
Means of
Auxinsconce.
Means of Auxins Type
2.7ef
2.5e
3.50b
3.8de
3.7d
IBA
0.8
4.6bcd
5.7abc
5.15abc
NAA
0.2
5.4abc
5.7abc
5.55ab
NAA
NAA
0.6
0.8
5.6abc
4.7bcd
6.5a
6.1ab
6.05a
5.40ab
4.53b
4.76a
Means of Media
5.00a
5.66a
according to Duncan's Multiple Range Test (Duncan, 1955).
3. Roots length (cm)
Data in Table (6) Fig (4,5)had been that the
maximum mean value of root lengths (4.04 cm)
were obtained due to modifying the ¼MS
medium, although, the shortest roots (3.23 cm)
were developed on the ½MS medium and it was
significantly increased with increasing auxin
concentrations. It is clear from the same Table
(6) shown that the mean value of type of auxins
were significant variances between IBA, NAA
and IAA on rooting lengths of photinia. The
longest root (4.62cm) was recorded in medium
having NAA and significant changes compared
with medium having IAA, and the longest
root(3.77)was recorded in medium having IBA.
In contrast, the mean result of auxin
concentration treatments showed that the highest
length of roots/ shootlets (5.05cm) was recorded
medium (0.6mgl-1), and the shortest roots
(2.81cm) were found on medium 0.2mgl-1IAA.
Comparable results were found by (Manisha, et.
al.2001) on Alnusnepalensis, and (Gad,et.
al.1999) on Khayaivorensis
The result of different media treatments and
its interactions with diverse types of auxin on the
three concentrations on photinia root length, that
the kinds of auxin and concentration have no
significant influence on this parameter. The
interactions between cultured media and
different types of auxins show that the ¼MS
medium with 0.6 mgl-1NAAenhanced rooting
and produced an average (5.29cm) compared to
all treatments having½MS medium. Conversely,
the lowest length of root was made (2.04cm)
with ½MS medium free auxin. The benefit of
NAA over other auxins has also been reported in
other plant species such as Rosa Damascena
(Boskabady, et. al.2011)Rosa grussanteplitz and
Rosa centifolia. (Mirza, et. al. 2011)
318
Table (6): Effect of salt strength of MS media and different types of auxin concentrations on average root length
(cm) of photinia after 8 weeks.
Root Length
Conce.
Means of
Means of Auxins
Mgl-1
½MS
¼MS
Auxinsconce
Type
Control
0
2.04g
2.28fg
IAA
0.2
2.31fg
3.32cdef
2.815ef
IAA
0.6
2.51efg
3.64cd
3.075e
3.02b
IAA
0.8
2.89defg
3.45cde
3.170ee
IBA
0.2
2.87defg
4.78ab
3.825cd
IBA
0.6
3.58cde
4.94ab
4.260bc
3.77ab
IBA
0.8
2.88defg
3.57cde
3.225de
NAA
0.2
4.21abc
5.12ab
4.665ab
NAA
0.6
4.81ab
5.29a
5.05a
4.62a
NAA
0.8
4.05bc
4.24abc
4.145bc
Means of Media
3.23b
4.04a
Auxins Type
Acclimatization Stage
The successful transfer of plantlets from
culture tubes to the soil is one of the most
essential steps in a sexual micro propagation
program of several plant species. The plantlets
were transferred from the in vitro conditions to
greenhouse location, Fig (6). After 8 weeks in
319
rooting medium, the rooted plantlets were
washed under tap water to remove the remains
of culture medium, which is a goal of
microorganism's attacks because of its carbon
source and agar content. The survival rate
reached to 85% and after 2 weeks the plantlets
transferred to greenhouse environments.
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APPLICATION METHOD OF POTASSIUM HUMATE ON GROWTH AND
YIELD OF GREEN ONION (ALLIUM CEPA L.)
Kurdistan Hassan Yousif
Faculty of Agriculture, School of Plant Production, Dept. of Horticulture, University of Duhok, Kurdistan Region –Iraq.
ABSTRACT:
The experiment was carried out during October 2011 at the vegetative research farm, Faculty of
Agriculture and Forestry, University of Duhok on onion (Allium cepa L. )., bulbs were planted in November 2011 .RandomizeCompleteBlock design (R.C.B.D.) was used in this experiment. Each treatment was
replicated three times with 10 plants per each. The factors included the following; two concentrations of
Potassium humate (0 and 1.8 gm.L-1) and application methods (foliar and fertigation),.Results showed that
Potassium humate caused significant increase in most of vegetative growth characteristics .The dual
interaction treatments among the tested factors at both fertilization methods especially by fertigation at
the high levels revealed significant increase in vegetative characters. and significant increase occurred in
the yield characters (bulbs weight, yield of a square meter kg.m2).
KEY WORDS: Potassium humate, Application Methods , Onion.
INTRODUCTION
o
nion (Allium cepaL.) belongs to the
family Amaryllidaceae which is one of
the most important mono-cotyledonuscrop. It is
an important crop in all condiment and used of
flavouring the food, both at mature and
immature bulb stages besides being used as salad
and pickles. It can be eaten as green leaves,
bulbs can be eaten as fresh and also can be used
in preparation of different dishes, Onions are the
fourth most consumed vegetable in Iraq after
tomatoes, potatoes and cucumbers., Onions are
good for human health(Shanmugavelu, 1990).
Onion
bulb
is
strongly
contracted
subterranean shoot with thickened, fleshy leaves
as food organs and the nutritional value of it per
100gm is recorded:Carbohydrates (11.0 g),
proteins (1.2 g), fiber (0.6 g), moisture (86.8 g)
and energy (38 cal). Apart from these, vitamins
like vitamin ‘A’ (0.012 mg), vitamin ‘C’ (11
mg), thiamine (0.08 mg), riboflavin (0.01 mg)
and niacin (0.2 mg), and the minerals like
phosphorus (39 mg), calcium (27 mg), sodium
(1.0 mg), iron (0.7 mg) and potassium (157 mg)
(Anonymous., 1978).Potassium humate contains
organic C (51-57%), N (4-6%) and P (0.02%). It
is believed that these acids improve crop yield
due to its capability of applying nitrogen and P
to the plants which added as a fertilizers in the
amount of 1-2kg.h-1 [Brannon, 1985].The
excessive use of agrochemical has polluted the
environment to a great extent and the food
produced under such a farm management may
not be safe or of a good quality therefore public
323
awareness of these problems has shafted the
approach toward some alternative measurement
(Shaxson, 2006).Organic fertilizers which
include humic materials are one of the natural
amendments which to increase the rate of
organic matter in soil associated with improving
the physical ,chemical and biological properties
of the soil and consequently improve plant
growth and development and the application of
humic substances affect the plant growth which
result in productive and fertile soil, and lead to
increase the water holding capacity of the soil., it
plays a pivot role in improving germination of
the plant the plant resistance against drought
stress, and reduces the requirement to the other
fertilizers(Phelps, 2000). It also increases crop
yield, soil aeration, and drainage can also be
enhanced byhumic, in addition to establishment
of desirable environment for the development of
microorganisms,potassium humate as organic
acid also play an important role which promote
physical and chemical charecters of the soil
through the vigore of these materials with soil
mineral (Matoroiev 2002). Potassium humate is
considered as an organic fertilizers it increase
product quality and plant tolerance to drought
stress salinity, heat cold, diseases and pest it
increase also potato tuber yield (Hassanet al.,
2008). Hafez (2004) reported that the application
of humic substances improved plant growth,
increase fruit yield and quality in squash plant
(cucrbbitapepo L.). Abd El- Aalea al. (2005)
revealed that application of potassium humate
with irrigation water (14.28L.ha) significantly
increased onion bulbs, dry weight of (Allium
cepa L.). Aays and Gulser (2005) studied
different doses of sulphur and humic acid on
yield components in squash (Spinaciaoleracea
L.), the application of humic acid increased the
total yield and the relation between doses of
sulphure and humic acid with the total yield was
found significant. Treatment of tomato seed with
0.01% potassium humate solution before
planting for 24hours lead to increasing the yield
about 20-25%., also pre-planting seed treatment
of cucumber by 0.01%potassium humate
solution for 24hours lead to increasing the yield
about 38%. Jarienet al. (2007).Abd-El Moueam.
(2013) carried out a field experiment on green
onion in Mousel., he appear that adding organic
fertilizers
improve
vegetative
growth
vharecters(leaves numbers, fresh weight and dry
weight) also he showed significant different in
yield charecters.
growth parameter which include (leaves length
(cm.) , No. of leaves.plant-1 , total chlorophyll
percentage (%), fresh and dry weight (gm)) and
also yield parameters (bulbs weight and yield
kg.h-1 ) was recorded.
The aim of this study was to improve the
vegetative growth and yield of green onion by
applying method potassium humate as soil and
foliar application on green onion plants.
3. RESULTS AND DISSCUTION:
2. MATERIALS AND METHODS
The experiment was carried out in
October2011 at the research farm, Faculty of
Agriculture and Forestry, University of
Duhokongreen onion (Allium cepa L. ),
TexsasGranocultivare., the land was ploughed
for two perpendicular lines and the soil was well
softed, thw whole area was divided in to the
three blocks, each experiment units consist of
three row of 2*1m . bulbs were planted at
distances of 25cm in October, 2011.at the third
upper par of the two side of the ridge the
fertilizer process by adding animal manure
before planting and the soil was irrigated then
the bulbs was planted. Randomize Completely
Block design (R.C.B.D.) was used in this study
that experiment included two factor, the first one
was the concentration of potassium humate (0
and 1.8 gm.L-1) and the other one represent the
method of application of it (foliar and
fertigation), after one month of planting the
plant were sprayed four times within ten
intervals day on the other hand all needed
agricultural and horticultural process was done
regularly during this study, the obtained data
was statistically analyzed by using
SAS
program, and the significant difference between
means was evaluated according to Duncan
multiple range test at 5 % level. both vegetative
EXPERIMENTAL MEASUREMENTS WERE
AS FOLLOWS:
1 –VEGETATIVE GROWTH
CHARACTERISTICS: a) Leaves length (cm). )
b) No. of leaves .plant-1.
c) Total chlorophyll content (SPAD).
d) Fresh weight of vegetative (gm).
e) Dry weight of vegetative (gm).
3- YIELD CHARACTERISTICS:a. Bulbs weight (gm).
b. Yield kg.m2.
3. 1. VEGETATIVE GROWTH CHARACTERS:
Table (1) Refer to the effect of potassium humate ,
its application method and their interaction on
vegetative growth characters, the data showed that there
was significant differences according to the method of
potassium huamte application in leaves length which
reaches (50.45cm) in fertigation compared with
44.5cm in foliar method in the same time there was
significant differences on leaves length as a result of the
concentration of potassium humate on the other hand
the effect of interaction between the treatment indicated
that there was significant difference in the application
method (fertigagation) and potassium humate at
(1.8gm.L-1 ) that gave high value of leves length (55.10
cm) compared with untreated one (control) 40.50cm
and 45.8cm in both method).
Concerning the leaves numbers .plant-1 it also refers
that the application method hawed significant increase
in fertigation application method which reaches (16.15
leaves plant-1) in fertigation compared with 14.05
leaves. Plant -1) in foliar method, in the same time there
was significant differences on leaves numbers as a
result of the concentration of potassium humate on the
other hand the effect of interaction between the
treatment indicated that there was significant difference
in the application method (fertigagation) and potassium
humate at (1.8gm.L-1 ) that gave high value of leaves
number.plant-1 (17.10 ) compared with untreated one
in foliar application method.
Table (1) also indicated the effect of potassium
humate , its application method and their interaction on
Chlorophyll content (SPAD), there was
significant
increase
in
chlorophyll
324
contentinfertigation application method (53.11)
compared with foliar application method
(51.71), in the same time there was significant
differences on chlorophyll content as results of
the adding 1.8gm.L-1 of potassium humate
56.55 compared with untreated once (control),
regarding the interactions the interaction
between fertigation application method and
potassium humat with 1.8gm.L-1 gave higher
value of chlorophyll % (57.18%) compared with
control in both application method.
Increasing vegetative growth by the
fertigationmethods of potassium humatemay be
attributed to the role of potassium humate on
improving the soil fertility and increasing the
availability
of
nutrient
elements
and
consequently increased plant growth . The plant
growth characters may give the clear indicators
on the size and dense of vegetative growth of
onion plants, [AL-Mokhtaret al ., 1991; Basset,
1986].Or may be due to the role of potassium
humatethat provides nutrient elements that share
in bio efficiency and then increasing the growth
[Abd-Almawgoodet al., 2007].
Regarding the fresh and dry weight.The
results indicated that fertigation had high
significant effects on fresh weight, than foliar.
The fresh weight was also significantly affected
by potassium humate levels. The plant treated
with 1.8gm.L-1 potassium humate had high fresh
weight (38.26gm) as compared with the
untreated plant (36.6gm). It also indicates that
fertigtionapplication method with potassium
humateat level (1.8gm.L-1) significantly affected
the fresh weight as compared with untreated
plant in foliar application method.
Regarding the dry weight there was
significant difference among the two application
method fertigation application method gave
hight value of fresh weight compared with foliar
application method, and there are significantly
affected by potassium humate levels,treating
plant with 1.8gm..L-1 potassium humateget high
dry weight (10.7gm) as compared with the
untreated plant. The application method ×
potassium humate interaction showed significant
effects on the dry weight of vegetative growth,
this may be as a result to the adding potassium
humate that helped the soil to ventilation which
permite the root respiration and, easily penetrate
in the soil then lead to increase the root growth
whose positively increases the vegetative growth
through water and nutrient absorption (Garcia et
al .,2008)The effect of potassium humate on
plant growth could be due to the presence of
325
plant growth regulators, which are produced by
increasing the activity of microbes such as fungi,
bacteria, yeasts, actinomycetes and algae that
potassium humate make the activation of
macroorganism better by adding nutrient
element and reduce the loss of it [Arancon et al
2004; Molivko, 2001].
The enhancing of the plant growth using
potassiumhumate had been reported to be due to
increasing nutrients uptake such as N, Ca, P, K,
(A.O.A.C. 2000).The beneficial effects of
potassium humate on plant growth may be
referred to its acting as source of plant growth
hormones(Abd El-Aalet al., 2005).
The leave length Increased due to the
application of potassium humate since the acid
have the ability to provide an acidic medium and
correlate with positive ions to form a complex
which is very important for trace elements
(micronutrients) as these micronutrients are
seized (cohered) tightly and protected from
precipitation by these compounds. The
potassium humate is also a source of Nitrogen
hence increasing the availability of nutrients
(Phelps, 2000).
3.2. YIELD CHARACTERISTICS:
Table (2) Refer to the effect of potassium humate ,
its application method and their interaction on yield
characters ( bulbs weight (gm) and yield kg.m2 ) the
data showed that there was significant differences
according to the application method of potassium
huamte in fertigation method significantly increased
which reaches (17.75gm) compared with 15.75gm in
foliar application method in the same time there was
significant differences on bulbs weight as a result of the
concentration of potassium humate on the other hand
the effect of interaction between the treatment indicated
that there was significant difference in the application
method (fertigagation) and potassium humate at
(1.8gm.L-1 ) that gave high weight of bulbs which
reached (21.1gm) compared with untreated plants
treatments.
Concerning the yield (kg.m2) of onion it also refers
that the application method showed significant increase
in
fertigation application method which reaches
(20.2kg. mt-2) in fertigation compared with 16.1 kg. mt-2
in foliar method, in the same time there was significant
increase on yield as a result of the concentration of
potassium humate on the other hand the effect of
interaction between the treatment indicated that there
was significant difference in the application method
(fertigagation) and potassium humate at (1.8gm.L-1)
that gave high value of yield kg.m2 (22 kg.m2)compared
with the other treatment..
Increasing yield and weight of bulbsmay be
attributed to that the potassium humates are
considered as an important source of organic
matter and their effects on yield and its
components could be through their enhancing
effect on increasing soil moisture holding
capacity, improving soil texture as well as
promoting the uptake of nutrients leading to
stimulation of plant growth, and consequently on
yield and its components (Zhangeet al ., 2003).
Ghonameet al. (2009) who state that the
highest yield were obtained when hot pepper
plants were sprayed with potassium humate as a
biostimulation. Rotenberg et al. (2005) reported
that addition of organic amendment (composts to
agricultural soils lead to improved soil quality
and reduce severity of crop diseases as well as
increased cucumber yield., and is in harmony
with[Azrami and Giglo, 2009]on cucumber;
[selim , et al 2009]
We can concluded from this study that ability
enhancing the growth and yield of green onion
by using some of organic fertilizer asalternative
to chemical fertilizers and their negative effect to
soil , environment and health.
Table (1): Effect of application methods and Potassium humate, and their interactions on the Vegetative growth
of green onion characters.
No. of leaves. Plant-
Leaves length (cm)
Application
Potassium humate
1
Mean effect
of
application
Application
0
1.8.L
Foliar
40.50b
48.6b
44.55b
Fertigation
45.8b
55.10a
50.45a
Mean effect
of P.H
43.15b
51.85a
Potassium humate
Mean effect
of
application
0
1.8gm.L
Foliar
14.02b
14.08b
14.05b
Fertigation
15.2ab
17.10a
16.15a
Mean effect
of P.H
14.61b
15.59a
Chlorophyll content(SPAD)
Potassium humate
Application
mean effect of application
0
1.8gm.L
Foliar
47.5b
55.93ab
51.71b
Fertiigation
Mean effect
of P.H
49.09b
57.18a
53.13a
48.29b
56.55a
Fresh weight (gm.)
Potassium humate
0
1.8gm.L
Mean effect
of
application
Foliar
32.4b
34.30b
33.35b
Fertigation
40.8a
42.23a
41.51a
Mean effect of
P.H
36.6b
38.26a
Application
Dry weight (gm.)
Potassium humate
0
1.8gm.L
Mean effect
of
application
Foliar
9.9c
10.2ab
10.05b
Fertigation
10.1b
11.2a
10.65a
Mean effect
of P.H
10b
10.7a
Application
Means within a column, row and their interactions followed with the same letters are not significantly different
from each other according to Duncan’s multiple range test at 5% level.
326
Table (2): Effect of application methods and potassium humate, and their interactions on the yield characters of
greenOnion.
Application
Bulbs weight gm.plant1
Potassium humate
0
1.8gm.L
Mean effect
of
application
Application
Yield kg. m2
Potassium humate
0
1.8gm.L
Mean
effect of
application
Foliar
13.5b
18ab
15.75b
Foliar
15c
17.2b
16.1b
Fertigation
14.4b
21.1a
17.75a
Fertigation
18.4ab
22a
20.2a
Mean effect of
P.H
13.95b
19.55a
Mean effect
of P.H
16.7b
19.6a
Means within a column, row and their interactions followed with the same letters are not significantly different
from each other according to Duncan’s multiple range test at 5% level.
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Gajewski. (2007). Effect of fertilizers on oil
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Diseases Plant Resistance. Ch. Agri .j .I :1516 . Russlan.
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and fertilization systems as the main
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Properties. Phelps Teknowledge.. 1424.
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.(2009). Beneficial effect of humic substances
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of carbon, water and soil :a different
perspective. Agron. Sustain. Dev. 26.9-19.
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328
HOMOGENEOUS PHOTOCATALYTIC DEGRADATION OF ACID
ALIZARIN BLACK USING HYDROGEN PEROXIDE
Haydar A. Mohammad Salim, Sabir Ayob Mohammad Salih and Sherwan M. Simo
Dept. of Chemistry, Faculty of Science, University of Zakho, Kurdistan Region-Iraq.
(Accepted for publication: November 11, 2014)
ABSTRACT
Photocatalytic degradation of acid alizarin black (AAB) dye (C.I. 21725) in aqueous solution was
investigated using UV light in the presence of hydrogen peroxide (H2O2) as a catalyst at different
operating conditions. The operating conditions were concentration of catalyst dosage (0.1, 0.2 and 0.3 mL
of 30 % H2O2), initial concentration of AAB dye (100, 150 and 200 mg/L) and pH (3.3, 6.84 and 10.8). It
was found that the increasing of catalyst concentration enhanced the dye decolourisation. Hydrogen
peroxide exerted positive effects on the AAB removal whilst the initial concentration of AAB negatively
affected its removal. It was also found that the removal efficiency of AAB increased with the pH value
close to neutral (pH 7), while a reversed trend was observed at acidic and basic medium.
KEY-WORDS: Photocatalytic, AOPs, catalyst, wastewater, H2O2.
INTRODUCTION
any modern techniques of treatment
have been used over the last few
decades to treat and purify water. Ozonation and
photolysis techniques, for example, have been
used successfully to degradate and reduce a
range of organic and biological pollutants (Rice
and Browning, 1981, Rice and Hoff, 1981,
Barakat et al., 2005). However, stronger oxidant
are produced (i.e. radicals) when these
techniques are combined with some additives
such as hydrogen peroxide and catalysts, and
these processes together with additives are called
Advanced Oxidation Processes (AOPs) (Glaze et
al., 1987). AOPs are typically based on redox
reactions as a result of gaining and losing
electrons by radicals and organic molecules,
respectively (Rice and Netzer, 1983). Among
AOPs, in recent years, homogeneous
photocatalysis methods have received a great
attention in degrading or reducing organic
pollutant (Al-Ekabi et al., 1991).
It is estimated that, in textile industries, more
than 10 % of the dye is lost during the process of
dyeing and discharged as effluent (Weber and
Stickney, 1993). Since the existence of small
quantities of dyes (even bellow 1 part per
million) is clearly visible, the discharge of those
coloured water pollutants in the environment is a
considerable non aesthetic pollution source.
Through hydrolysis, oxidation or other chemical
reactions taking place in the phase of
wastewater, wastes of dyes can also produce
dangerous by-products and eutrophication
(Zollinger, 1991, Tang et al., 1997). Therefore,
dye effluents decolourisation has received
M
329
increasing attention. In liquid phase, addition of
hydrogen peroxide (H2O2), as oxidant, is an
accepted technique to accelerate photocatalytic
oxidation. For photocatalytic oxidation of water
phase, the hydrogen peroxide effect was
extensively investigated. Addition of moderate
H2O2 concentration was found to be significantly
accelerate degradation of dyes and aromatic
compounds (Barakat et al., 2005, Mart'yanov et
al., 1997, Balcioglu and Inel, 1996, Dionysiou et
al., 2004). This study outlines the results
achieved for the degradation of acid alizarin
black (AAB) in the presence of H2O2. Various
operating parameters were studied in this
research including initial AAB concentration,
amount of H2O2 and pH.
METHODOLOGY
Materials
Acid alizarin black (AAB) was purchased
from Hopkin and Williams LTD, UK and used
as received. A stock solution of AAB (1000
mg/L) was prepared on a daily basis in distilled
water and other concentrations (100, 150 and
200 mg/L) were prepared by dilution the stock
solution of AAB. The prepared stock solution
was covered by aluminum foil and kept in dark.
Hydrogen peroxide (H2O2) was purchased from
ALPHA, India and used as received without
further treatment. UV lamp with 254 nm (12
watt) was purchased from SEMTEC, China.
Experimental procedures
A closed semi-batch reactor, as shown in Fig.
1, was used in this study. A 500 mL, at a specific
concentration, of the AAB solution was charged
into the reactor, this solution prepared from the
stock solution by dilution. 30 % of H2O2 at a
spesific volume (0.1, 0.2 and 0.3 mL) was added
to the AAB solution. The volume of the reactor
was 600 mL. It is made from PYREX glass and
fitted with a sample port. The reactor was
equipped with a plunging tube in which a
SEMTEC 12 watt lamp was placed horizontally.
A glass syringe with 5 mL volume was used, at a
specific schedule, to collect samples. The pH
values (3.23, 6.84, and 10.8) of these solutions
were adjusted using 0.1M of NaOH and 0.1M of
HNO3. The pH of the solution was determined
using pH meter (EUTECH, Malaysia). Visible
spectrophotometer (Jenway, 6700) was used to
analyse the collected samples.
Fig. (1) Photocatalytic reactor
The effect of H2O2 concentration on AAB
decolourisation was studied and the results are
shown in Fig.2. The figure shows the
decolourisation of AAB solution in the presence
of H2O2. The other experiment conditions were
kept constant (AAB concentration was set to 100
mg/L, pH 6.84, and room temperature 24oC).
Various volume of H2O2 (0.1, 0.2 and 0.3 mL)
Effect of initial H2O2 concentration
were used. Fig. 2 also shows how the increasing
catalyst amount increases the removal efficiency.
After 30 min reaction time, the decolourisation
rate of AAB increased when the amount of H2O2
increased, due to the formation of highest
amount OH● radicals when more H2O2 volume
was used.
330
Fig. (2) AAB removal by using different amount of H2O2 at 30 min ([AAB]o = 100 mg/L, Temperature= 24 oC,
Volume = 500 mL, pH= 6.84)
After 5 min of the reaction (see Fig. 3), the
removal percentage of AAB increased and
reached 59 %, after using 0.1 mL of H2O2.
However, the removal percentage enhanced and
reached 81 % and 84.5 % when the volume of
H2O2 increased to 0.2 and 0.3 mL, respectively.
The removal efficiency of AAB was recorded
the highest removal percentage when 0.3 mL of
(H2O2)wasused.
Fig. (3) AAB removal percentage at various amount of H2O2 at 5 min ([AAB]o = 100 mg/L, Temperature= 24
o
C, Volume = 500 mL, pH= 6.84)
331
Effect of initial AAB concentration
The effect of initial AAB concentration on
the decolourisation rate of AAB was studied
using UV light in the presence of H2O2. The
results show that increasing the initial
concentration
of
AAB
reduced
the
decolourisation rate (see Fig. 4). The rate
constant, k', was found to decrease linearly with
increased initial AAB concentration. When AAB
concentration increased from 100 to 200 mg/L,
the decolourisation percentage of AAB at 5 min
reaction time decreased from 81 % to 21 %,
respectively (see Fig. 5). The dye loss, according
to the following equation, was observed as a
function of time and data were fitted to a pseudo
first order rate model (Bali et al., 2004, Esplugas
et al., 2002).
The plot of ln (C/Co) versus time gives a
straight line with k' = - slope (k', as seen from
figure, depends on the H2O2 concentration), so
the reaction follows a pseudo-first order scheme.
Table 1 and Table 2 show the effect of dye
concentration and amount of H2O2 on rate
constant.
Table (1): Effect of AAB concentration on a pseudofirst order kinetic for the degradation of AAB
[AAB] (mg/L)
k' (min-1)
R2
100
0.2483
0.9608
150
0.1511
0.9808
200
0.0844
0.994
Table (2): Effect of H2O2 amount on a pseudo-first
order kinetic for the degradation of AAB
H2O2 Volume
(mL)
k' (min-1)
R2
0.1
0.1953
0.984
0.2
0.2327
0.960
8
0.3
0.3052
0.986
5
Fig. (4): Effect of initial AAB concentration on solution decolourisation at 60 min (Temperature= 23 oC,
Volume= 500 mL, pH= 6.84)
332
Fig. (5) AAB removal percentage at various concentrations of AAB at 5 min (Temperature= 23 oC,
Volume = 500 mL, pH= 6.84)
Effect of pH
The process of AAB removal is higher at
pH close to 7 compared to AAB removal in
acidic and basic medium. As shown in Fig. 6,
the removal percentage improved from 94 to 97
% when pH changed from 3.3 to 6.84,
respectively. However, the improvement
decreased from 97 to 95.4 % when pH increased
from 6.84 to 10.8, respectively.
Fig. (6) Effect of pH on AAB removal percentage at 30 min (Temperature= 23 oC, Volume = 500 mL)
333
contaminants-synergism and inhibition in a
continuous-mode photocatalytic reactor.
Applied Catalysis B: Environmental, 50,
259-269.
-ESPLUGAS, S., GIMÉNEZ, J., CONTRERAS, S.,
PASCUAL, E. & RODRı́GUEZ, M. 2002.
Comparison of different advanced oxidation
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1987. The Chemistry of Water Treatment
Processes Involving Ozone, Hydrogen
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Oxidation of Methyl Viologen in an
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CONCLUSIONS
Photocatalytic degradation, in the presence of H2O2,
of AAB under different operating conditions
was studied in this research. The operating
conditions were initial dye concentration,
amount of H2O2 and pH. The most effective
improvements on the degradation of AAB
were
recorded
with
initial
AAB
concentration of 100 mg/L. It was also
found that the increasing of H2O2 quantity
enhance the reaction rate of AAB
decolourisation. The removal efficiency of
AAB was favourable in the neutral medium
more than the acidic and basic medium. The
removal percentage reached the maximum
value at pH close to 7.
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Materials, 114, 159-166.
-BARAKAT, M. A., TSENG, J. M. & HUANG, C.
P. 2005. Hydrogen peroxide-assisted
photocatalytic oxidation of phenolic
compounds.
Applied
Catalysis
B:
Environmental, 59, 99-104.
-DIONYSIOU, D. D., SUIDAN, M. T., BAUDIN,
I. & LAÎNÉ, J. M. 2004. Effect of hydrogen
peroxide on the destruction of organic
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@ómbè@ŽôïèbäìŠ@ŽõŠò‡äbè@bÙŽî‹i@ (AAB, C.I. 21725)@•òŠ@ŽôåîŠaïÜó÷@Žô’‹m@bÌbîíi@bäìí›ÙŽïm@a‡ŽïåïÜíØóÄ@ŽôÄ†
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335
PHOTOCATALYTIC DEGREDATION OF ACID ALIZARIN BLACK USING
POWDER AND NANOPARTICLES OF TITANIUM DIOXIDE
Haydar A. Mohammad Salim, Sherwan M. Simo and Neewar A. Yaseen
Dept. of Chemistry, Faculty of Science, University of Zakho, Kurdistan Region-Iraq.
(Accepted for publication: June 22, 2014)
ABSTRACT
Photocatalytic degradation of acid alizarin black (AAB) dye (C.I. 21725) in aqueous solution was
investigated using UV light in the presence of powder of titanium dioxide (P- TiO2) and nanoparticles of
titanium dioxide (N- TiO2) as a catalyst. The operating conditions were catalyst dosage (10 and 20 mg/L)
and initial concentration of AAB dye (10 and 20 mg/L). It was found that the increasing of catalyst
concentration enhanced the dye decolourisation. Both catalysts exerted positive effects on the AAB
removal whilst the initial concentration of AAB negatively affected its removal.
KEYWORDS: Photocatalytic, AOPs, catalyst, wastewater, nanoparticles.
INTRODUCTION
any modern techniques of treatment
have been used over the last few
decades for treat and purify water. Ozonation
and photolysis techniques, for example, have
been used successfully to degrade and reduce a
range of organic and biological pollutants (Rice
and Hoff 1981; Rice and Browning 1981).
However, stronger oxidant are produced (i.e.
radicals) when these techniques are combined
with some additives such as hydrogen peroxide
and catalysts, and these processes together with
additives are called Advanced Oxidation
Processes (AOPs) (Glaze, Kang et al. 1987).
AOPs are typically based on redox reactions as a
result of gaining and losing electrons by radicals
and organic molecules, respectively (Rice and
Netzer 1983). Among AOPs, in recent years,
heterogeneous photocatalysis methods have
received a great attention in degrading or
reducing
organic
pollutant
(Al-Ekabi,
Safarzadeh-Amiri et al. 1991).
It is estimated that, in textile industries, more
than 10% of the dye is lost during the process of
dyeing and discharged as effluent (Weber and
Stickney 1993). Since the existence of small
quantities of dyes (even bellow 1 part per
million) is clearly visible, the discharge of those
coloured water pollutants in the environment is a
considerable non aesthetic pollution source.
Through hydrolysis, oxidation or other chemical
reactions taking place in the phase of
wastewater, wastes of dyes can also produce
dangerous by-products and eutrophication
(Zollinger 1991; Tang, Zhang et al. 1997).
Therefore, dye effluents decolourisation has
received increasing attention. Using TiO2 as a
photocatalyst,
among
heterogeneous
M
photocatalysis, appears as the most emerging
destructive method for decolourisation of dye
effluents (Ollis and Al-Ekabi 1993). By using
irradiation and solar technology, different kinds
of dyes have been successfully reduced or
degraded in a batch scale (Neppolian, Sakthivel
et al. 1998; Wang 2000; Zhu, Wang et al. 2000).
This study outlines the results achieved for the
degradation of acid alizarin black (AAB) in the
presence of both powder TiO2 (P-TiO2) and
nanoparticle of TiO2 (N-TiO2). Various
operating parameters were studied in this
research including initial concentration of AAB
and catalyst dose.
METHODOLOGY
Materials
Acid alizarin black (AAB) was purchased
from Hopkin and Williams LTD, UK and used
as received. A stock solution of AAB (100mg/L)
was prepared on a daily basis in distilled water
and other concentrations (10 and 20 mg/L) were
prepared by dilution the stock solution of AAB.
The prepared stock solution was covered and
kept in dark. Titanium dioxide (TiO2), with
particle size of 45 μm, was purchased from
ALPHA CHEMIKA, India; and nanoparticles of
TiO2, with particle size of 21 nm, was purchased
from Sigma-Aldrich, UK, and used as received
without further treatment. UV lamp with 254 nm
(12 watt) was purchased from SEMTEC, China.
Experimental procedures
A closed semi-batch reactor, as shown in Fig.
1, was used in this study. A 500 mL of the AAB
solution at a specific concentration was charged
into the reactor, this solution prepared from the
stock solution by dilution. The volume of the
336
reactor was 600 mL. It is made from PYREX
glass and fitted with a sample port. The reactor
was equipped with a plunging tube in which a
SEMTEC 12 watt lamp was placed horizontally.
A glass syringe with 5 mL volume was used, at a
specific schedule, to collect samples. A
centrifuge was used for 10 min to separate
titanium dioxide from the solution before
analysis. Visible spectrophotometer (Jenway,
6700) was used to analyse the collected samples.
Fig. (1) Photocatalytic reactor
Effect of initial TiO2 concentration
The effect of TiO2 concentration on AAB
decolourisation was studied and the results are
shown in Fig.2. The figure shows the
decolourisation of AAB solution in the presence
of both P-TiO2 and N-TiO2. The other
experiment conditions were kept constant (AAB
concentration was set to 10 mg/L, pH 6.84, and
337
room temperature 22oC). The catalyst
concentrations used were 10 and 20 mg/L for
both catalysts. Fig. 2 also shows how the
increasing catalyst concentration increases the
removal efficiency. After 60 min reaction time,
the decolourisation rate of AAB increased when
concentration of both catalysts increased. It was
found that the rate of decolourisation enhanced
when N-TiO2 was used instead of P-TiO2.
Fig. (2) AAB removal by using different concentrations of catalyst at 60 min ([AAB]o = 10 mg/L, Temperature=
22 oC, Volume = 500 mL, pH= 6.84)
The removal percentage of AAB increased
and reached 77 % and 87 % after using 10 and
20 mg/L of P-TiO2, respectively (see Fig. 3).
However, the removal percentage enhanced and
reached 90 % and 94 % when 10 and 20 mg/L of
N-TiO2 used, respectively. The removal
efficiency of AAB was recorded the lowest
removal percentage when 10 mg/L of P-TiO2
used.
Fig. (3) AAB removal percentage at various concentrations of catalyst at 60 min ([AABo = 10 mg/L,
Temperature= 22 oC, Volume = 500 mL, pH= 6.84)
338
Effect of initial AAB concentration
The effect of initial concentration of AAB on
the decolourisation rate of AAB using UV light
in the presence of P-TiO2 and N-TiO2 was
studied. The results show that increasing the
initial concentration of AAB reduced the
decolourisation rate (see Fig. 4). The
decolourisation percentage of AAB in the
presence of P-TiO2 at 60 min reaction time
decreased from 77 % to 74 % when AAB
concentration increased from 10 to 20 mg/L,
respectively (see Fig. 5). However, the
decolourisation percentage in the presence of NTiO2 decreased from 90 % to 87 %.
Fig. (4) Effect of initial AAB concentration on solution decolourisation at 60 min (Temperature= 22 oC, Volume
= 500 mL, pH= 6.84)
(90 %)
(87 %)
(77 %)
(74 %)
Fig. (5) AAB removal percentage at various concentrations of AAB at 60 min (Temperature= 22 oC,
Volume = 500 mL, pH= 6.84)
339
CONCLUSIONS
Photocatalytic degradation of acid alizarin
black (AAB) in the presence of powder of TiO2
(P-TiO2) and nanoparticles of TiO2 (N-TiO2),
under different operating conditions, was studied
in this research. The operating conditions were
catalyst dosage and initial concentration of
AAB. The most effective improvements on the
degradation of AAB were recorded when NTiO2 was used. It was found that the increasing
of catalyst concentration enhance the reaction
rate of AAB decolourisation. It is also found that
the removal percentage of AAB reached the
maximum value when 10 mg/L of AAB used
instead 20 mg/L.
REFERENCES
- Al-Ekabi, H., A. Safarzadeh-Amiri, et al. (1991).
"Advanced technology for water purification
by
heterogeneous
photocatalysis."
International Journal of Environment and
Pollution 1(1-2): 125-136.
- Glaze, W. H., J. W. Kang, et al. (1987). "The
Chemistry of Water Treatment Processes
Involving Ozone, Hydrogen Peroxide and
Ultraviolet Radiat." International Ozone
Association 9: 335-352.
- Neppolian, B., S. Sakthivel, et al. (1998).
Photocatalytic degradation of textile dye
commonly used in cotton fabrics. Studies in
Surface Science and Catalysis. T. S. R. P. Rao
and G. M. Dhar, Elsevier. Volume 113: 329335.
- Ollis, D. F. and H. Al-Ekabi (1993). Photocatalytic
purification and treatment of water and air:
proceedings of the 1st International
Conference
on
TiO2
Photocatalytic
Purification and Treatment of Water and Air,
London, Ontario, Canada, 8-13 November,
1992, Elsevier.
- Rice, E. W. and J. C. Hoff (1981). "Inactivation of
Giardia lamblia cysts by ultraviolet
irradiation." Appl Environ Microbiol 42(3):
546-547.
- Rice, R. G. and M. E. Browning (1981). Ozone
treatment of industrial wastewater. Park
Ridge, N.J., Noyes Data Corp.
-Rice, R. G. and A. Netzer (1983). "Handbook of
Ozone Technology and Applications, Vol 1,
Rice,Rg, Netzer,A." Journal American Water
Works Association 75(5): 64-64.
- Tang, W. Z., Z. Zhang, et al. (1997). "TiO2/UV
photodegradation of azo dyes in aqueous
solutions." Environmental Technology 18(1):
1-12.
- Wang, Y. (2000). "Solar photocatalytic degradation
of eight commercial dyes in TiO2 suspension."
Water Research 34(3): 990-994.
- Weber, E. J. and V. C. Stickney (1993). "Hydrolysis
kinetics of Reactive Blue 19-Vinyl Sulfone."
Water Research 27(1): 63-67.
- Zhu, C., L. Wang, et al. (2000). "Photocatalytic
degradation of AZO dyes by supported TiO2 +
UV in aqueous solution." Chemosphere 41(3):
303-309.
- Zollinger, H. (1991). Color chemistry: syntheses,
properties, and applications of organic dyes
and pigments, VCH.
340
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341
THE HYDROLOGICAL COMPARISIONS BETWEEN THE CHALK
AQUIFER AND THE HOLDERNESS GLACIAL TILL OF SMALL
CATCHWATER DRAIN CATCHMENT IN HOLDERNESS, ENGLAND.
Ramadhan Haji Sulaiman Zaidky
Petroleum Engineering Department, School1of Engineering, University of Zakho, Kurdistan Region of Iraq.
(Accepted for publication: October 29, 2014)
Abstract:
The research programme was about the hydrology of this part of Holderness, particularly in respect of
the interactions between superficial and deeper hydrological system, in order that the long term findings
from the Catchwater Drain catchment might be more usefully applied to the solution of hydrological
problems, including agricultural water supplies, land drainage, flooding and need for irrigation.
There has been no attempt, however, in previous work to quantify the hydrological relationships
between the clay and sand/gravel areas of the catchment or even to determine precisely the geographical
extent of the sand/gravel layers and lenses.
In the early studies, the assumption made was that the catchment was hydrologically watertight.
This research has resolved a number of outstanding uncertainties which have raisin during the long
period of operation of the Catchwater Drain catchment and that it has shed valuable new light on the
hydrology, not only of the Catchwater Drain catchment but also of the glacial till of Holderness.
It is to be hoped that the improved understanding which has thereby resulted will be of value in the
interpretation of the hydrological behaviour of extensive, similar areas elsewhere.
Introduction
Catchwater Drain Catchment was one of the
few experimental areas in Britain in which
glacial till hydrology has been intensively
studied which is situated at the east side of the
Holderness plain of North Humberside, England
(Figure 1). The Catchwater Drain catchment,
some 15.5 km sq. in area. The topography of the
catchment mirrors that of the wide area of
Holderness in which the boulder clay plain rises
imperceptibility towards the coast in the east and
the Yorkshire Wolds in the west and north.
Relief in the catchment ranges from about 25 m
O.D. in the north-east to about 7.5 m O.D. at the
exit of the Catchwater Drain in the south-west,
and consequently the majority of the slopes are
quite gentle.
Inevitably the heterogeneous character of the
glacial till provides a hydrogeological context
which has important repercussions on the
storage and movement of water within the
catchment. Of these hydrogeological influences
the most important are likely to be variations of
pore-size and pore-size distribution, especially
between the sediment types; the existence of
hard pan layers which will impede vertical water
movement;
and
the
existence
and
interconnectivity of lenses or bodies of coarser
material within the more widespread clay.
Data Collection and Instrumentations.
The hydrological data from the Catchwater
Drain catchment and from the clay and
sand/gravel sub-catchment, (Figure 2), are
compared with hydrological data for the Chalk
aquifer. Particular attention is paid to data on the
Chalk piezometric surface and to comparisons of
the variation of that surface and variations of
ground water level and stream flow which take
place in the Catchwater Drain catchment. These
comparisons are made over different time-scales
and for different data intervals. The research
programme is concerned with "conventional"
hydrological data such as ground water levels
and discharge data. It is important to emphasise,
however, that the present study has generated a
very large amount of data. Some of this derives
from the processing of raw data, e.g. water level,
stream flow, and data collected as part of the
ongoing catchment experiment, but
342
343
344
not processed before the begging of this
research programme. This applies to all data
collected between 1979 and 1986. Also some
important new data has been collected from the
other authorities such as the Yorkshire Water
Authority, the Soil Survey of England and Wales
and the Institute of Hydrology. Finally, entirely
new data have been collected and processed as
part of this research programme.
In this way it is hoped to compare, interpret
and demonstrate the relationship, between the
varying hydrological conditions in the
underlying Chalk aquifer and hydrological
conditions within the till catchment of the
Catchwater Drain.
The hydrology of glacial till.
Because glacial till or boulder clay comprises
such
a
heterogeneous
collection
of
morphological features and particle-sizes
relatively massive clays on the one hand and
lenses and pockets of sand/gravel or even
coarser material on the other, its hydrology is
equally non-uniform and therefore difficult to
characterise. With hydraulic conductivities
typically in the range 8.64x10-3 to 8.64x10-5
cm/day (Freeze and Cherry, 1979), boulder clay
forms some of the most extensive shallow
aquitards in North America and Europe. In the
sand/gravel deposits, however, such as those
found in the study area, where hydraulic
conductivities may range from 20.41 to 114.62
cm/day (Bonell, 1971) transmission rates are
much higher and water storage is much more
dynamic . What limited interest there has been
in the hydrology of glacial till areas has,
therefore, tended to concentrate on the localised
potential ground water resources of the coarser
materials (c.f. Todd, 1980; Ehler and Grieger,
1983; Kowalski and Janiak, 1986; Michel, 1986;
Johnson and Williams, 1987).
Certainly, there has been little attempt to
consider glacial drift hydrology on a scale or
even to take it seriously at all in Britain, where
till deposits are in any case shallow, and where,
from the point of view of water resources, the
dynamic hydrological system in the coarser
materials are very small both spatially and in
terms of saturated thicknesses.
Hydrologically, these patches of sand and
gravel and other lighter material can be regarded,
345
in comparison with the widespread boulder clay,
as islands of extremely high permeability where
the average of 65.36 cm/day compares with 1.27
cm/day in the clay (Bonell, 1971). These
deposits therefore constitute potentially excellent
aquifers for ground water storage although
severe limitations are likely to be imposed by
their limited extent and depth. In this respect
particular significance would attach to
sand/gravel deposits which rest directly on
Chalk. This does occur just outside the
catchment at Routh Carr (Chadha, 1988) see
(figure 3), and in northern Lincolnshire (Lloyd
( 1980) see (Figure 4), 1980).
Comparison between ground water levels in
the Chalk Aquifer and in the sand and clay
areas and Catchwater Drain discharge (19781988).
In order to explore the relationships between
some aspects of the hydrology of the Chalk and
the Catchwater Drain catchment, monthly
instantaneous ground water levels for the Chalk
and for the sand and clay areas, together with
monthly instantaneous values of discharge at the
catchment outlet, are examined for the period
from 1978 to 1988 (excluding 1979 for which
the relevant data are incomplete). The Chalk
aquifer ground water levels were recorded by the
Yorkshire water Authority (1988) in a well at
Hornsea, which is located some 5.0 km to the
north of Great Hatfield. Hydrological data,
plotted monthly for two contrasting years, are
shown in Figure 5 and 6. The summer period of
1987 was drier than the average and as Figure 5
illustrates, there was a steady decline in both
ground water and discharge values from April to
September. The total April-September runoff
from the Catchwater Drain catchment in 1980
was virtually identical (38.5 mm compared with
38.2 mm in 1987). It will be clear from Figure 6,
however, that catchment runoff in 1980 was
much lower during the early part of the summer
and rose to a marked but short-lived peak in
August as a result of the occurrence of heavy
rainfall. Although this peak is reflected in the
graphs of ground water level for both the Chalk
and the Great Hatfield sandy area, those ground
water responses were very muted. This is to be
expected, since the discharge values
346
347
348
for the entire catchment would have
incorporated a significant quick flow element.
The pattern in the remaining years for which
data are available was intermediate between
these two extremes, showing a close similarity in
all years in the variations of ground water level
between the Chalk aquifer and the sand area and
a less obvious similarity in some years between
the catchment discharge variations and the
ground water hydrographs. Period averages of
the instantaneous values are shown for each
month in Figure 7 and these confirm the general
relationships outlined above.
Further exploration of the relationships
between ground water conditions in the Chalk
aquifer and in the sand and clay areas of the
Catchwater Drain was carried out using simple
regression analysis. The scattergrams in Figure 8
and 9 show the regression relationships between
ground water conditions in the Chalk and in the
Great Hatfield sandy area and the regression
statistics for each year of the data period are set
out in Table 1.
Despite contrasting hydrological conditions
in 1986 (when total April-September runoff
from the Catchwater Drain catchment was 86.3
349
mm) and 1987 (when total April-September
runoff was 38.2 mm), the scattergrams in Figure
8 illustrate a strong and highly significant
relationship between Chalk and sand ground
water conditions. Indeed, in 7 of the 10 years for
which data are presented, R2 values exceeded
0.85 and in 8 of the years the relationships were
significant at the 99% level. This generally
significant relationship over data period is
reflected in Figure 9 which is a composite
scattergram for all 10 years.
Relevant comparative data for the Chalk
aquifer and the clay area at Hatfield Wood Farm
were only available for six years, i.e. 1982-1988.
Data for 1987 were incomplete and have
therefore been excluded. Table 2 summarises the
statistical data for these years and a composite
scattergram is shown in Figure 10. The
relationship is clearly weak and indeed in only
one year does the R2 value exceed 0.5 and the
level of significance exceed 95%, i.e. in most
years there was in fact no identifiable
relationship between the Chalk ground water
levels and those in the clay areas of the
Catchwater Drain catchment.
350
351
352
353
Comparision between hydrological conditions
in the Chalk Aquifer and in the Catchwater
Drain catchment during the study period
(1987-1988).
Figure 11 illustrates, for the 1987-1988
period, further aspects of the relationship
between ground water levels in the Chalk aquifer
and stream flow and ground water levels in the
sand and clay areas in the Great Hatfield and
South Field sub-catchments. Although the
cessation of stream flow and the drying out of
the tube-wells with increasing desiccation at the
clay transect severely restricts the usefulness of
such comparisons, it is clear from Figure 11 and
Table 3 that there was in both years, but
especially in 1987, a close similarity between the
recession of Chalk ground water levels and the
recessions of stream flow and ground water level
plotted for the Great Hatfield sub-catchment. In
1988 it will be noted that, although the Chalk
and sand ground water levels peaked in May and
declined steadily thereafter, stream flow from
the sand area peaked in June. A substantially
different relationship appeared to exist in the
clay area since, in the earlier part of the 1988 dry
season, i.e. April to June, when ground water
levels and stream flows were recorded in the
clay area, their variations with time did not
resemble very closely the ground water
variations within the Chalk aquifer.
Conclusion
The data interpreted as indicating a direct
hydrological relationship between the Chalk
aquifer and the Great Hatfield sandy area.
Certainly, their pattern of ground water
fluctuations was similar. In the much larger clay
area of the catchment, however, the pattern of
ground water fluctuations did not resemble
closely those in the underlying Chalk aquifer
during the early part of the summer. In addition,
later in the summer, falling ground water levels
in the clay areas led to drying-up of the open
ditches. Clearly these apparent hydrological
relationships would be expected if the Great
Hatfield, and perhaps other, bodies of sand and
gravel are indeed in hydraulic continuity with
the Chalk aquifer providing strong evidence that
there is an upward movement of ground water
from the Chalk into the sand area at great
Hatfield, either because they rest directly upon
the Chalk aquifer, similar situations occur also
in northern Lincolnshire (Lloyd, 1980 ), and in
Norfolk, as shown in Figure 4, where permeable
crag deposits glacial sand rest directly on the
underlying Chalk aquifer, or because of the
presence of very localized permeable drift
materials which permit hydraulic continuity
through the till to the surface.
354
355
REFERENCE
Bomell .m. (1971). An in investigatiom of the
movement and fluctuation of ground water in
a small glacial catchment in halderness , east
yorlcshirt, ph.d.thesis. university hull.
Chadha.D.S.(1988). Personal Commnnicotion
Ehlers, J. And J. Hrieger (1983). Ground Water
Chemistry In The Hamburg Region, IAHS
Pupl. No. 156: 285-95.
Free Ze , R.A. And I.A. Jerry (1979). Gnund Water,
Printice – Hall, Englewood Clidds, N.J. ,
Goupp.
Foster, S.S.D. And A.S. Robertson (1977). Evolution
Of A Semiconfined Chalk Aqnifer In East
Anglia, Proc. I.C.E.,63
Johansson, P. O. (1987). Estimation of ground water
recharge in sandy till with two different
methods using ground water level fluctuations,
J. Hydrol., 90: 183-98.
kowasaki, J. and Z. Janiak (1986). Ground water
changes in the urban area of wroclaw in the
period 1874-1947, IAHS Publ., No. 156: 4557.
Lloyd, J. W. (1980). The influence of Pleistocene
deposits on the hydrogeology of major British
aquifers, J. Inst. Water Eng. Sci., 34: 346-56.
Michel, F. A. (1986). Hydrogeology of the Central
Mackenzie Valley aquifer. J. Hydrol., 85: 379405.
Tood, D. K. (`980). Ground Water Hydrology, John
Wiley and Sons, New York.
356
‫‪Journal of University of Zakho, Vol. 2(A) , No.2, Pp 342-357, 2014‬‬
‫جياوازيێن ھايدرولوجى دناڤبهرا عهمبارێن ئاڤێ تهباشيرى و بهر رهخێ گێچێ ھولدرينس يێ چوونا‬
‫ئاڤ گرتى ل كومكهرهك بچيك ل ھولدرينسى ‪ /‬انگلترا‬
‫پوخته‪:‬‬
‫پروگرامێ ڤهكولينێ ل سهر ھايدرولوجيا ئهڤێ پارچا ھولدرينسى بتايبهتى ژاليێ كارتێكرنێن ناڤبهرا‬
‫سيستهمێ ھايدرولوجى ێن كيروسهرڤه ‪ .‬ژبهر كو ديتنێن دير ژ گرتنا چوونا ئاڤێ ژ ئاڤا كومكرى‬
‫باشتر بھێته بكارھينان بو شروڤهكرنا ئاريشێن ھايدرولوجى ێن بخوڤه گريديت وهكو دابينكرنا ئاڤا‬
‫رووهكى ‪ ,‬رهواندنا عهردى الفاو ‪ ,‬و پێتڤين بو ئاڤدانێ‪.‬‬
‫ل كارێ بهرێ چ ھهول نهھاتهدان بو قهبارهكرنا پهيوهنديێن ھايدرولوجى ناڤبهرا جھێن قور‬
‫)تهقن( و جھێن خيز ‪ /‬بهرخشك يێ كومكهرێن ئاڤێ يانكو حهتابھيرى دياركرنا پێڤهچونا جوگرافى يا‬
‫تهخهوچينێن خيز‪ /‬بهرخشكا‪.‬‬
‫ل خواندنێن زى ھاتينهكرن ‪ ,‬وا دانا ھاتيه دانان ئهو بو كو ئهو كومكهرنا ژاليێ ھايدرولوجى ئاڤ‬
‫گرتى بو ‪ .‬ئهڤێ ڤهكولينێ ژمارهك ژ گومانێ ديار ئهوێن دوركهفتين ل كردارێن دهم درێژ يا چوونا ئاڤا‬
‫گرتى ل كومكهرى و ئهڤێ روناھى يهكى ببن دا رێژا ل سهر زانينا ئاڤێ ) ھايدرولوجى( نهك بتنێ يا‬
‫چوونا ئاڤا گرتى ل كومكهرى بهلێ ھهروهسا يا بهر رهخێ گێچێ ھولدرينسى‪.‬‬
‫ئومێد ئهوهى كو تێگهھشتنێن باش يێن ھاتين ئهنجام دا دێ ببھابن ل لێكدانا رهفتارێ ھايدرولوجى‬
‫لجھێن وهكى ڤى جھێ ل ھهر دهڤهرهكێ ھهبيت ‪.‬‬
‫‪@Í–@Êávn¾@ëŠí—a@ëbï¾a@ÒîŠb—nÜ@óî‡ïÝ§a@ïäŠ‡Üíè@pbj‹mì@ÝÙÜa@çíÙà@ça‚@μi@óïuíÜìŠ‡ïa@pbäŠbÕ¾a‬‬
‫‪@@NaÝÙäa@L@ïäŠ‡Üíè@óÕåà@ôÐ‬‬
‫‪@@Z˜ƒÝ¾a‬‬
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‫‪@@êmb‡Èì@ô—äO@Þà‹Üa@pbÕjÜ@ÖïÔ†@ÞÙ“i@Àa‹Í§a@†a‡nàýa@‡î‡¥@ôny@ìa@Êávn¾a@a‰@ô—äO@Þà‹Üaì‬‬
‫‪@HwatertightIóáÙ«@ëbïà@æÈ@òŠbjÈ@oäbØ@êÉávn¾a@ëbï¾a@ça@ôÝÈ@óïåjà@óÕibÜa@pbaŠ‡Üa@ôÐ@aÐýa@çbØ‬‬
‫‪@@NóïuíÜìŠ‡ïè@óÕî‹i@óÉávnà‬‬
‫‪@ëbï¾a@Òî‹—m@pbïÝáÉÜ@ÞîíÜa@paÑÜa@ßþ‚@p‡Èb—m@‡Ô@ônÜaì@òjÙÜa@pbéj“Üa@æà@a†‡È@Þy@‡Ô@szjÜa@a‰è‬‬
‫‪@ëbï¾a@ Òî‹—m@ óïÑïØ@ ôÐ@ ÂÕÐ@ ïÜ@ óïuíÜìŠ‡ïa@ óaŠ‡Üa@ ôÐ@ öí›Üa@ æà@ a‡îà@ ÂïÝ—m@ ¶a@ p†a@ ônÜaì@ êÉávn¾a‬‬
‫‪@@N@ïäŠ‡Üíè@óÕå¾@óî‡ïÝ§a@pbjÜa@ôÐ@b›îa@æÙÜì@êÉávn¾a‬‬
‫‪@òrØ@õ‹‚a@Ö bå¾@ðuíÜìŠ‡ïa@ÛíÝÜa@Ñm@ôÐ@óáïÔ@ìˆ@çíÙï@szjÜa@a‰è@w÷bnä@ôÐ@ŒÉ¾a@âéÑÜa@ça@Þàü¾a@æà‬‬
‫‪@@ïäŠ‡Üíè@óÕå¾@óéib“nà‬‬
‫‪357‬‬
NUMERICAL INVESTIGATION OF PRANDTL NUMBER EFFECTS ON
THE NATURAL CONVECTION HEAT TRANSFER FROM CIRCULAR
CYLINDER IN AN ENCLOSED ENCLOSURE
Omar Mohammed Ali
Department of Refrigeration and Air Conditioning, Technical Institute of Zakho, Kurdistan - Region, Iraq.
(Accepted for publication: August 12, 2014)
ABSTRACT
In the present work, the natural convection heat transfer from horizontal circular cylinder situated in
a square enclosure is investigated numerically. The work investigates the effect of Prandtl numbers on the
flow and heat transfer characteristics. The study uses different Prandtl numbers (0.03, 0.7, 7, and 50),
different Raylieh numbers (104, 105, and 106) and different enclosure width to cylinder diameter ratios
W/D (1.667, 2.5 and 5). The work included the solution of the governing equations in the vorticity-stream
function formulation which were transformed into body fitted coordinate system. The transformations are
based initially on algebraic grid generation and elliptic grid generation to map the physical domain
between the heated horizontal cylinder and the enclosure into a computational domain. The disecritization
equation system are solved by using finite difference method. The code build using Fortran 90 to execute
the numerical algorithm.
The results were compared with previous numerical results, which showed good agreement. The effect
of Prandtl number variation on the average Nusselt numbers, flow patterns and isotherms with different
Raylieh numbers and enclosure width ratios were investigated. The flow patterns and temperature
distributions are presented by means of streamlines and isotherms, respectively. The results show that the
streamlines and isotherms for Pr=0.03 are unique and differ from those of other higher Prandtl numbers
for all enclosure widths and Ra≥105. The streamlines and isotherms for Pr≥0.7 are nearly similar and
independent of Prandtl number. The same behaviors as streamlines and isotherms occur with Nusselt
number for lower and higher values of Prandtl numbers with all ratios of enclosure width to cylinder
diameter.
KEYWORDS: Heat Transfer, Circular Cylinder, Square Enclosure, Numerical.
@
NOMENCLATURE
Symbol
Nu
D
di,j
H
J
K
P
P
Pr
Q
R
Ra
T
Definition
Average Nusselt
number, (h.D/k).
Cylinder diameter.
Source term in the
general equation,
eqn. (12).
Convective heat
transfer coefficient.
Jacobian.
Thermal conductivity
of the air.
Pressure.
Coordinate control
function.
Prandtl number,
(/).
Coordinate control
function.
maximum absolute
residual value.
Raylieh number,
(gTD3/).
Time.
Unit
T
U
m
V
W
W
W/m2.
C
W/m.
C
N/m2
X
X
y
Y
T
seconds

Temperature.
Velocity in xdirection.
Velocity in ydirection.
Enclosure Width.
Relaxation factor.
Horizontal direction
in physical domain.
Dimensionless
horizontal direction
in physical domain.
Vertical direction in
physical domain.
Dimensionless
vertical direction in
physical domain.
Greek Symbols
Difference between
cylinder surface
temperature
and environmental
temperature.
Viscosity of the air.
C
m/s
m/s
cm
m
m
C
kg/m.s
358











S

X
Y

D

T

Coefficient of
thermal expansion.
Vertical direction in
computational
domain.
Horizontal direction
in computational
domain.
Dimensionless
stream function.
Vorticity.
Dimensionless
vorticity.
Kinematic viscosity.
Thermal diffusivity.
Dimensionless
temperature.
Dependent variable.
Stream Function.
Subscript
Cylinder surface.
Environment.
Derivative in xdirection.
Derivative in ydirection.
Derivative in direction.
Circular cylinder
diameter.
Stream function.
Temperature.
Vorticity
1/C
1/s
m2/s
m2/s
1/sec.
INTRODUCTION
T
he flow and thermal fields in enclosed
space are of great importance due to their
wide applications such as in solar collectorreceivers, cooling of electronic equipment,
aircraft cabin insulation, thermal storage system,
and cooling systems in nuclear reactors, etc. A
large number of literatures were published in the
past decades. The effects of Prandtl number on
natural convection heat transfer are of great
interest. The Prandtl number is defined as the
ratio of the kinematic viscosity of a fluid to its

thermal diffusivity, = , which could vary over

several orders of magnitude from 10-2 to 105 for
common fluids. For example, the family of
liquid metals has a small Prandtl number (Pr<101
) and on the contrary, the Prandtl number of
engine oils is extremely large (Pr>102).
Moreover, air and many other gases, water, and
the family of hydrocarbons occupy the
intermediate range
(10-1<Pr<102) of the entire
359
spectrum. Since the Prandtl number serves as a
measure of the viscous diffusion rate relative to
the thermal diffusion rate, fluids with different
Prandtl numbers could have quite different
natural convective flow and heat transfer
characteristics in enclosures or confined layers,
Zi-Tao Yu et al., 2010.
Natural convective heat transfer in horizontal
annuli between two concentric circular cylinders
has been well studied. A comprehensive review
was presented by Kuehn and Goldstein, 1976.
Comparatively, little work has been done on
natural convective heat transfer in more complex
annuli such as the problem considered in this
study. A few publications were involved in the
experimental study. Ekundayo et al., 1998,
studied natural convection in horizontal annulus
between an outer square cylinder and an inner
circular cylinder. In their study, a cylindrical
heater with diameter of 9.5 mm was placed at
different locations within a 350 mm×350 mm
square-sectioned cold enclosure. The aspect
ratio, i.e., the side length of the outer square
cylinder over the diameter of the inner cylinder,
is 36.84, which is large. It was found that the
maximum steady-state rate of natural convection
occurred when the heater was located parallel to
and near a vertical wall. A few publications were
also found for numerical investigations.
Moukalled and Acharya, 1996, studied
numerically natural convective heat transfer
from a heated horizontal cylinder placed
concentrically inside a square enclosure. The
governing equations in their work are solved in a
body-fitted coordinate system using a control
volume-based numerical procedure. Shu et. al.,
2002, studied the natural convection in a
concentric annulus between a cold outer square
cylinder and a heated inner circular cylinder is
simulated using the differential quadrature
(DQ)method. The authors found that both the
aspect ratio and the Rayleigh number are critical
to the patterns of flow and thermal fields. They
suggests that a critical aspect ratio may exist at
high Rayleigh number to distinguish the flow
and thermal patterns. However, although there
are a great many papers that have documented
the Prandtl number effects on natural convection
in enclosures. Koca et al., 2007, numerically
investigated the Prandtl number effect on natural
convection in a horizontal triangular enclosure
that is locally heated on the bottom. Zi-Tao Yu
et al., 2010, studied parametrically the effects of
Prandtl number on laminar natural convection
heat transfer in a horizontal equilateral triangular
cylinder with a coaxial circular cylinder is
conducted. The Prandtl number is varied over a
wide range from 10-2 to 105, which corresponds
to a variety of working fluids. The governing
equations with the Boussinesq approximation for
buoyancy are iteratively solved using the finite
volume approach. It is shown that the flow
patterns and temperature distributions are unique
for low-Prandtl number fluids (Pr≤0.1), and are
nearly independent of Prandtl number when
Pr≥0.7. In addition, the inclination angle of the
triangular enclosure is found to noticeably affect
the variations of the local Nusselt number, and to
have insignificant influence on the average
Nusselt numbers for low Rayleigh numbers
when Pr≥0.7.
The present work deals with numerical
investigation of natural convection heat transfer
from circular horizontal cylinder situated in an
enclosed square enclosure. The effect of Prandtl
numbers with different Raylieh numbers Ra, and
enclosure width on the Nusselt number will be
investigated. The behaviors of the flow and
temperature distribution will also be investigated
to gain insight in the effect of studied variables
on the flow hydrodynamics and thermal
behavior.
MATHEMATICAL FORMLATION
The governing equations of the flow between
the heated horizontal cylinder and the enclosure,
that shown in figure (1), were based on the
assumptions that the flow is Boussinesq,
incompressible, no internal heat sources, laminar
flow, and two-dimensional, Ali, 2008.
T
Ts
Te
W
W
Figure (1): Configuration of cylinder-enclosure combination
The governing equations include the equation
of continuity, momentum and the energy
equation, Bejan and Kraus, 2003. These
equations are presented below:
u v

0
x y
(1)
v
v
v
1  p   v


u
v


t
x
y
  y  x   x 
  v 



 y   y 
(3)
The energy equation is:
The x –momentum equation is:
1  p   u 
u
u
u


u
v


  x  x   x 
t
x
y
  u 
  g T


 y   y 
The y –momentum equation is:
 T
T
T
u
v

t

x
 y

 c 
(2)


 y
 T 
 k

  y


 
x


 T 
 k

  x  (4)
Where  is the laminar viscosity.
360
With Boussinesq approximations, the density
is constant for all terms in the governing
equations except for the buoyancy force term
that the density is a linear function of the
temperature.
   o 1   T 
(5)
Where  is the coefficient of thermal expansion.
The stream function () and vorticity () in
the governing equations are defined as follows,
Anderson, 1995 and Petrovic, 1996:


u
, v
y
x
(6)
v u

x y

Or     V

The cylinder diameter D is the characteristic
length in the problem. By using the above
parameters,
The
governing
equations
(8)-(10)
transformed to the following general form in the
computational space:
  1        
a   
   
      b    d 
  t J         
(12)
Where  is any dependent variable.
The governing equations represented by
interchanging the dependent variable  for three
governing equations as follow
(7)
the governing equations for laminar flow
become:

a
b
d

0
1


1

T
1
 k
Energy Equation:
          2  2 




 t  y  x  x  y   x 2  y 2 

t
(8)
Momentum Equation:
1
J
  2  2 
      



 Pr  2 
t  y  x  x  y
 y 2 
x

 Ra Pr
x
(9)
 
x
2

2
 
 y2

x
D
,
 
t
D
2
Y
,
y
D
(10)

,

,

U

uD

V
,
D 2
,


(11)
361



 y 
 
0
represents the unsteady term.
       

   
   is the convective term.
      

 b  

is the diffusion term.
Grid Generation
In the stream function-vorticity formulation,
there is a reduction in the number of equations to
be solved in the - formulation, and the
troublesome pressure terms are eliminated in the
- approach.
The dimensionless variables in the above
equations are defined as:
X 
y  
In addition, d  is the source term.
Continuity Equation:
2
g
J
vD

,
  
c  
The algebraic grid generation method is used
to generate an initial computational grid points.
The elliptic partial differential equations that
used are Poisson equations:
 xx   yy  P  ,  
 xx   yy  Q  , 
(13a)
(13b)
Interchanging dependent and independent
variables for equations (13a, and b), gives:
 x  2 x   x 


J 2 P x  Q x  0
 y   2 y    y 


J 2 P y   Q y  0
(14a)
(14b)
(16)
Where   x2  y2 ;   x x  y y ;
a P  a E  a W  a N  a S  a Po
  x2  y2
The resulting algebraic equation is solved
using alternating direction method ADI in two
sweeps; the first sweep, the equations are solved
implicitly in -direction and explicitly in direction. The second sweep, the equations are
solved implicitly in -direction and explicit in direction. In first sweep, the implicit
discretization equation in -direction is solved
by using Cyclic TriDiagonal Matrix Algorithm
(CTDMA) because of its cyclic boundary
conditions. In second sweep, the implicit
discretization equation in -direction is solved
by using TriDiagonal Matrix Algorithm
(TDMA).
The solution of the stream function equation
was obtained using Successive Over-Relaxation
method (SOR).
The initial conditions of the flow between
heated cylinder and vented enclosure are:
The coordinate control functions P and Q
may be chosen to influence the structure of the
grid, Thomas et. al., 1980. The solution of
Poisson equation and Laplace equation are
obtained using Successive over Relaxation (SOR)
method with relaxation factor value equal to 1.4,
Hoffman, 1989 and Thompson, 1985.
The transformation of the physical domain
into computational domain using elliptic grid
generation is shown in figure (2).
=0,  = 0,  = 0
(17)
For t = 0
The temperature boundary condition of the
cylinder surface assumed as constant.
Figure (2) Transformations of the physical
domains into computational domains using
elliptic grid generation.
at enclosure wall
(18a)
Using 2nd order difference equation, the
temperature at the enclosure surface becomes:
4
3
1
3
 i ,m   i ,m 1   i , m  2
Method Of Solution
In the present study, the conversion of the
governing integro-differential equations into
algebraic equations, amenable to solution by a
digital computer, is achieved by the use of a
Finite Volume based Finite Difference method,
Ferziger, 2002.
To avoid the instability of the central
differencing scheme (second order for
convective term) at high Peclet number (Cell
Reynolds Number) and an inaccuracy of the
upwind differencing scheme (first order for
convective term) the hybrid scheme is used. The
method is hybrid of the central differencing
scheme and the upwind differencing scheme.
a P  P  a E  E  aW W  a N  N  a S  S  a Po  Po 


0
 m

a M i 1, j 1  i 1, j 1  i 1, j 1  i 1, j 1  d i , j
(15)
(18b)
Vorticity boundary conditions, Roache, 1982,
are
 
2
J2
 i,m   i,m1 
at enclosure wall
(19a)

2
J2
 i,1  i,2 
at cylinder surface
(19b)
The stream function of the cylinder is
assumed as zero because the cylinder is a
continuous solid surface and no matter enters
into it or leaves from it. The stream function of
the enclosure is assumed as constant.
The Nu is a nondimensional heat transfer
coefficient that calculated in the following
manner:
362
Nu 

J
2

0

d

(20a)
The derivative of the nondimensional
temperature is calculated using the following
formula, Fletcher, 1988 :
 

1

     
n   const . J 
J 


(20b)
 = 0 at cylinder surface
A computer program in (Fortran 90) was built
to execute the numerical algorithm which is
mentioned above; it is general for a natural
convection from heated cylinder situated in an
enclosure.
In the present study, different fluids were
used as the working fluids and the Prandtl
number ranged between (0.03-50). The
numerical work deals with natural convection
heat transfer from circular horizontal cylinder
when housed in an enclosed square enclosure.
The cases for three different aspect ratios W/D
=5, 2.5 and 1.67 and Rayleigh numbers of 104,
105, and 106 were studied.
After numerical discretization by the Hybrid
method, the resultant algebraic equations are
solved by the ADI method. The convergence
criteria are chosen as RT<10-6, R<10-6 and
R<10-6 for T,  and  respectively. When all
the three criteria are satisfied, the convergent
results are subsequently obtained.
Stability And Grid Independency Study
0.7. Three time steps are chosen with values
1×10-4, 5×10-4, 5×10-6. The maximum difference
between the values of Nu with different time
steps is 2%. The numerical method becomes
unstable with time step 10-4 at Pr0.7, therefore;
the time step of the method decreases with
changing the value of the Prandtl number.
The grid-independence of numerical results is
studied for the case with Ra=104, and 105, W/D
=2.5, Pr = 0.7. The three mesh sizes of 96×25,
128×45, and 192×50 are used to do gridindependence study. It is noted that the total
number of grid points for the above three mesh
sizes is 2425, 5805, and 9650 respectively.
Numerical experiments showed that when the
mesh size is above 96×45, the computed Nu
remain the same. The same accuracy is not
obtainable with W/D=5 and high Raylieh
numbers, therefore; the mesh size 12845 is
used in the present study for all cases.
Validation Test
The code build using Fortran 90 to execute
the numerical algorithm. To test the code
validation, the natural convection problem for a
low temperature outer square enclosure and high
temperature inner circular cylinder was tested.
The calculations of average Nusselt numbers and
maximum stream function max for the test case
are compared with the benchmarks values by
Moukalled and Acharya, 1996, for different
values of the enclosure width to cylinder
diameter ratios (W/D=1.667, 2.5, and 5) with
Rayleigh numbers Ra=104 and 105 as given in
table (1). From table 1, it can be seen that the
present results generally agree well with those of
Moukalled and Acharya, 1996.
The stability of the numerical method is
investigated for the case Ra=105, W/D=2.5, Pr =
Table (1): Comparisons of Nusselt numbers and maximum stream function with Previous data.
max
L/D
5.0
2.5
1.67
5.0
2.5
1.67
363
Ra
104
105
Present
2.45
3.182
5.22
10.10
8.176
4.8644
Moukalled and
Acharya, 1996
2.08
3.24
5.4
10.15
8.38
5.10
Present
1.7427
0.9584
0.4274
3.889
4.93
6.23
Moukalled and
Acharya, 1996
1.71
0.97
0.49
3.825
5.08
6.212
Flow Patterns and Isotherms For W/D=1.667
Flow Patterns
The numerical solutions for four Prandtl
numbers were obtained. The Prandtl number
values are: Pr = 0.03, 0.7, 7, and 50 will be
presented herein. At W/D = 1.677, the Prandtl
number effects on natural convection flow
patterns, demonstrated by streamlines, are shown
in Fig. (3). The circular cylinder diameter is
relatively large and the physical domain between
the circular cylinder and the enclosure is small.
The maximum stream function value varies
between max=0.48 at Ra=104 to max=8.8 at
Ra=106 for low Prandtl number (Pr=0.03),
while; it varies between max=0.464 at Ra=104
to max =22.1 at Ra=106 for higher Prandtl
numbers (Pr≥0.7). The maximum stream
function value for Pr=0.03 decreases because the
convective flow is dominated by the inertia flow
rather than viscous flow. The flow is
symmetrical about the vertical line through the
center of the circular cylinder for all cases. The
flow patterns for Pr = 0.03, Ra≥105 are unique
and completely different from those for higher
Prandtl numbers Pr≥0.7. At Ra=104, the flow
circulation is very weak and the maximum
stream function value is small. The streamlines
for all Prandtl numbers are similar and
independent of Prandtl numbers with little
difference in the streamlines for Pr=0.03. The
flow patterns appear as a curved kidney-shaped
dual-kernel eddy. At Ra=105, the flow
circulation becomes stronger than Ra=104 and
the stream function value increases. The flow
patterns for Pr=0.03 are unique and completely
different from those of higher Prandtl numbers.
Multiple eddies appear for Pr=0.03, while;
appear two tiny eddies near the vertical center
line in addition to the eddies around the cylinder
for higher Prandtl numbers Pr≥0.7. The flow
patterns of Pr=0.03 becomes slightly oscillatory
in the lower part of the annulus and the steadystate solution is not available because the
convective flow is dominated by the inertia force
rather than the viscous force. The flow appear as
four eddies, two of them are nearly circularshaped near the upper corners of the enclosure,
and other two eddies appear as nearly ellipticalshaped. It is noted the appearance of two tiny
eddies near the vertical center line. The nearly
circular-shaped eddies have little densely
packed. For Pr≥0.7, the flow patterns are similar
and independent of Prandtl number. At Ra=106,
the maximum stream function values increase
for all Prandtl numbers. The flow circulation
become stronger than other Raylieh numbers.
The number of eddies increases to six eddies,
four of them near the corners of the enclosure
and two eddies inside the tiny eddies. The eddies
become more densely packed. The flow patterns
for Pr=0.7 differ from those of other higher
Prandtl numbers. The eddies move upward and
the stagnant area become more. A single large
kernel eddy appears for Pr=0.7 rather than the
small dual kernel eddies for Pr≥7. The flow
patterns cover most of the physical space
between the cylinder and the enclosure and the
area is very small.
364
(a)
(b)
(c)
Pr=0.03
Pr=0.7
Pr=7
Pr=50
Figure (3) Effect of Prandtl number on streamlines at W/D = 1.67 and (a) Ra = 104, (b) Ra = 105, (c) Ra = 106.
Temperature Fields:
The characteristics of the temperature
distributions are presented by means of
isotherms in figure (4). The same arrangements
as flow patterns are displayed in the figure with
same Prandtl numbers and Raylieh numbers. The
isotherms are symmetrical about the vertical line
through the center of the circular cylinder.
At Ra=104, the isotherms are similar and
independent of Prandtl number for all Prandtl
numbers. The isotherms display as rings around
the cylinder. The shape of the isotherms ensure
that the mode of heat transfer is pure conduction
and the effect of the convection is very low. At
Ra=105, the temperature distributions for higher
Prandtl numbers are similar and independent of
Prandtl number. They have small distortions
below the cylinder due to the effect of the
365
convection heat transfer. The isotherms for
Pr=0.03 are unique and differ from other Prandtl
numbers. The distortion of the isotherms become
more around the cylinder due to the effect of the
convection heat transfer. At Ra=106, the effect of
natural convection on the heat transfer increases.
For Pr≥7, a thermal plume impinging to the top
of the enclosure. Pair of thermal plumes appear
on the top of the cylinder with about 45 from
the vertical center line. For Pr=0.7, a thermal
plume with reverse direction appears on the top
of the cylinder, in addition to two thermal
plumes display with about 30 from the vertical
center line. A thermal stratification (horizontal
and flat isotherms) are formed for Pr≥0.7. The
isotherms become more flat in the middle region
below the cylinder for Pr≥7.
(a)
(b)
(c)
Pr=0.03
Pr=0.7
Pr=7
Pr=50
Figure (4) Effect of Prandtl number on isotherms at W/D = 1.67 and (a) Ra = 104, (b) Ra = 105, (c) Ra = 106.
Flow Patterns and Isotherms For W/D=2.5
Flow Patterns
The flow patterns for Prandtl number Pr =
0.03, 0.7, 7, and 50 with Raylieh numbers
Ra=104, 105, 106 are presented in figure(5). The
same arrangements that uses for the streamlines
of the case W/D = 1.677 are presented herein. At
W/D=2.5, the Prandtl number effects on natural
convection flow patterns are demonstrated by
streamlines, as shown in Fig.(5). The circular
cylinder diameter reduces and the physical
domain between the circular cylinder and the
enclosure enlarges. The maximum stream
function value varies between max=0.87 at
Ra=104 to max=12.73 at Ra=106 for low Prandtl
number (Pr=0.03), while; it varies between
max=1.739 at Ra=104 to max=24.78 at Ra=106
for higher Prandtl numbers (Pr≥0.7). As the case
W/D=1.67, the maximum stream function value
for Pr=0.03 decreases because the convective
flow is dominated by the inertia flow rather than
viscous flow. For all Prandtl numbers and
Raylieh numbers, the flow is symmetrical about
the vertical line through the center of the circular
cylinder. The flow patterns for Pr= 0.03 and all
Raylieh numbers are unique and completely
different from those for higher Prandtl numbers
Pr≥0.7. At Ra=104, the flow circulation is weak,
it become stronger than the flow circulation for
W/D=1.67. The maximum stream function value
is small. The streamlines for higher Prandtl
numbers Pr≥0.7 are similar and independent of
Prandtl numbers. The flow patterns appear as a
curved kidney-shaped contain dual kernel eddies
with different sizes. The flow patterns for
Pr=0.03 are differ from those of higher Prandtl
numbers Pr≥0.7. The streamlines move to the
sides and the stagnant area enlarges.
At Ra=105, the strength of the flow
circulation becomes more, and the value of
stream function increases. The flow patterns for
higher Prandtl numbers Pr≥0.7 are nearly similar
and independent of Prandtl number. A single
366
small kernel eddy appears rather than the dual
kernel eddies for Ra=104 and Pr≥0.7. The size of
kernel eddy for Pr=0.7 is larger than those for
Pr≥7. The streamlines near the bottom enclosure
wall move upward and the stagnant area
becomes more. The flow patterns for Pr=0.03 are
unique and completely different from those of
higher Prandtl numbers. There exist four nearly
circular-shaped eddies, which are slightly
asymmetrical about y-axis through the cylinder.
The flow becomes slightly oscillatory in the
lower part of the annulus and the steady-state
solution is not available because the convective
flow is dominated by the inertia force rather than
the viscous force. The eddies near the upper
corners have more densely packed than those
near the bottom corners. Two tiny eddies appear
near vertical center line on the bottom enclosure
wall. As Raylieh number increases to Ra=106,
the flow becomes stronger and the maximum
stream function increases for all Prandtl
numbers. The flow are symmetrical about the
vertical center line. The streamlines for Pr≥7 are
similar and independent of the Prandtl number.
The streamlines near the bottom enclosure wall
move more and more to the upward that lead to
an increase in the stagnant area. The streamlines
near the upper enclosure wall are horizontal and
flat. The size of the kernel becomes more. The
flow patterns for Pr=0.7 are similar to those for
Pr≥7, except the streamlines near the upper
enclosure wall and upper corners appear as
curvature. The appearance of multiple eddies
continue for Pr=0.03 and Ra=106. The eddies
become less densely packed, specially for lower
eddies. Pair of tiny eddies appear near the
vertical center line on the top of the cylinder.
(a)
(b)
(c)
Pr=0.03
Pr=0.7
Pr=7
Pr=50
Figure (5) Effect of Prandtl number on streamlines at W/D = 2.5 and (a) Ra = 104, (b) Ra = 105, (c) Ra
= 106.
367
Temperature Fields:
The temperature distributions for W/D=2.5
are presented by means of isotherms in figure
(6). The same arrangements as flow patterns are
displayed in the figure with same Prandtl
numbers and Raylieh numbers. The isotherms
are symmetrical about the vertical line through
the center of the circular cylinder. As Raylieh
number increases, the thermal boundary layer
becomes thinner and thinner.
numbers. The mode of heat transfer is the
conduction with very little effect of convection
heat transfer. The isotherms display as circles
around the cylinder. As Raylieh number
increases
to
Ra=105,
the
temperature
distributions are similar and independent of
Prandtl number for Pr≥0.7. The isotherms
distorts below the cylinder due to the effect of
the convection heat transfer. A thermal plume
appear on the top of the cylinder. The isotherms
for Pr≥7 are horizontal and flat near the lower
enclosure wall with very little distortion in the
isotherms for Pr=0.7 at this region. The
isotherms for Pr=0.03 are unique and differ from
those of other Prandtl numbers. The distortion of
the isotherms become more around the cylinder
due to the effect of the convection heat transfer.
At Ra=106, the convection becomes the
dominant mode of heat transfer. The isotherms
for Pr≥0.7 are similar with slightly difference for
Pr=0.7. A thermal plume impinging on the top of
the enclosure. For Pr≥7, the isotherms near the
bottom enclosure wall become more flat and
horizontal as compared with those for Pr=0.7.
The thermal stratification (horizontal and flat
isotherms) are formed at this region. Two
thermal plumes displayed on the top of the
cylinder with about 45 from the vertical center
line. The curvature of the these plumes are less
than those for Pr=0.7.
(a)
(b)
(c)
Pr=0.03
Pr=0.7
Pr=7
Pr=50
Figure (6) Effect of Prandtl number on isotherms at W/D = 2.5 and (a) Ra = 104, (b) Ra = 105, (c) Ra = 106.
368
Flow Patterns and Isotherms For W/D=5
Flow Patterns
The flow patterns for W/D=5 are presented
herein by means of streamlines with Pr = 0.03,
0.7, 7, and 50 and Ra=104, 105, 106 as shown in
figure (7). The same arrangements that use for
the streamlines of the cases W/D = 1.677, and
2.5 are displayed herein. At W/D=5, the circular
cylinder diameter is relatively small and the
physical domain between the circular cylinder
and the enclosure enlarges. The maximum
stream function value varies between
max=1.215 at Ra=104 to max=14.14 at Ra=106
for low Prandtl number (Pr=0.03), while; it
varies between max=1.81 at Ra=104 to
max=25.2 at Ra=106 for higher Prandtl numbers
(Pr≥0.7). The maximum stream function value
for Pr=0.03 decreases because the convective
flow is dominated by the inertia flow rather than
viscous flow. The flow patterns for Pr= 0.03 and
all Raylieh numbers are unique and completely
different from those for higher Prandtl numbers
Pr≥0.7. At Ra=104, the flow circulation is weak,
it become stronger than the flow circulation for
previous cases. The streamlines for higher
Prandtl numbers Pr≥0.7 are nearly similar and
slightly independent of Prandtl numbers. The
flow patterns appear as a curved kidney-shaped
contain one kernel eddy for each side. The flow
patterns for Pr=0.03 are differ from those of
higher Prandtl numbers Pr≥0.7. The flow is
asymmetrical about vertical line through circular
cylinder. The flow circulation is weak. The
369
stagnation area is large and the streamlines are
less densely packed from those of other Prandtl
numbers.
At Ra=105, the flow circulation becomes
stronger, which, the value of maximum stream
function increases. For Pr≥0.7, the streamlines
are nearly similar and independent of Prandtl
number, and the flow is symmetrical about
vertical line through circular cylinder. The
streamlines near the bottom of the enclosure wall
move upward toward the cylinder and the
stagnant area becomes more. The flow patterns
for Pr=0.03 are unique and completely different
from those for Pr≥0.7 for the same reason as
mentioned in the previous cases W/D=1.67 and
2.5. The flow become slightly asymmetrical
about y-axis through the cylinder. The shape of
the streamlines seem as rings attached with a
plume eddy below these circles. Multiple tiny
eddies display near the enclosure walls. At
Ra=106, the flow circulation becomes stronger
and the maximum stream function increases for
all Prandtl numbers. For Pr=0.03, the nearly
circular-shaped eddies become more densely
packed and the attached plumes direct to the
sides of the enclosure. The number of tiny eddies
around the enclosure increase. For Pr≥7, the
streamlines near the bottom of the enclosure wall
move upward to reach the bottom of the cylinder
and the stagnant area becomes more and more.
For Pr=0.7, the flow appear as nearly beanshaped eddies, which are symmetrical, are
formed. Two small eddies display near the
center line at the lower enclosure wall.
(a)
(b)
(c)
Pr=0.03
Pr=0.7
Pr=7
Pr=50
Figure (7) Effect of Prandtl number on streamlines at W/D = 5 and (a) Ra = 104, (b) Ra = 105, (c) Ra = 106.
Temperature Fields:
The temperature distributions for W/D=5 are
presented by means of isotherms in figure (8).
The same arrangements as flow patterns are
displayed in the figure with same Prandtl
numbers and Raylieh numbers. The isotherms
are symmetrical about the y-axis line through the
center of the circular cylinder. As Raylieh
number increases, the thermal boundary layer
becomes thinner and thinner.
numbers. The mode of heat transfer is the
conduction with little contribution of convection
heat transfer. The isotherms display as nearly
elliptical-shaped around the cylinder. As Raylieh
number increases to Ra=105, the isotherms
distort below the cylinder for higher Prandtl
numbers Pr≥0.7 due to the effect of the
convection heat transfer. The temperature
distributions appear as nearly similar and
independent of Prandtl number. A thermal plume
appear on the top of the cylinder. Two thermal
plumes displayed on the top of the cylinder with
about 60 from the y-axis center line. The
those of other Prandtl numbers. The distortion of
the isotherms become more around the cylinder
due to the effect of the convection heat transfer.
The thermal plume, which appears above top of
the cylinder, is thicker than that for Pr≥0.7. Two
thermal plumes are formed near the sides of the
cylinder with reverse direction. At Ra=106, the
convection becomes the dominant mode of the
heat transfer. The isotherms for Pr≥0.7 are
different. For Pr=0.7, a thinner thermal plume
impinging on the top of the enclosure. Two
plumes appear on top of the inner circular
cylinder with about 60◦ from the vertical centre
line. The isotherms below the cylinder are wavy.
For Pr≥7, the thermal plume above top of the
cylinder becomes thinner and increases its
length.
The isotherms below the cylinder
become more flat and horizontal as compared
with those for Pr=0.7. For Pr=0.03, the isotherms
distort more and more, and appear as wavy
around the cylinder. The thickness of the thermal
plume above top of the cylinder is thicker from
those for Pr≥0.7.
370
(a)
(b)
(c)
Pr=0.03
Pr=0.7
Pr=7
Pr=50
Figure (8) Effect of Prandtl number on isotherms at W/D = 5 and (a) Ra = 104, (b) Ra = 105, (c) Ra = 106.
Overall heat transfer and correlations
The average Nusselt number is chosen as the
measure to investigate the heat transfer from the
circular cylinder. The effect of Prandtl numbers
on the average Nusselt numbers with Ra=104,
105, and 105 for enclosure width to the cylinder
ratios W/D=1.67, 2.5 and 5 are presented in
figures (9, 10, 11). The Prandtl numbers in the
present study are:0.03, 0.7, 7, 50. The Nusselt
number increases with increasing the Raylieh
number for all Prandtl numbers and all enclosure
width to cylinder diameter ratios W/D. At
Ra=104, the Nusselt number do not change with
the variation of the Prandtl number and the
curves appear nearly flat because the conduction
is the dominant mode of the heat transfer for all
enclosure width to cylinder diameter ratios. At
Ra=105, the relation between the Prandtl number
and Nusselt number differ with the variation of
the W/D. For W/D=1.67, figure (9), the Nusselt
number independent of the Prandtl number,
therefore; the curve is nearly horizontal and flat
line. For W/D=2.5, figure (10), the Nusselt
number slightly decreases for Pr=0.03, but the
Nusselt number is independent of Prandtl
371
number for Pr≥0.7, which means the relation
between the Nusselt number and the Prandtl
number is flat (constant value) for Pr≥0.7. The
same behavior occurs for W/D=5, figure (11),
the Nusselt number is independent of Prandtl
number and slightly constant for Pr≥0.7. The
Nusselt number of Pr=0.03 is lower than that for
Pr≥0.7 by 1. At Ra=106 and W/D=1.67, figure
(9), the maximum Nusselt number value obtains
at Pr=0.7, and decreases as Prandtl number
increases to Pr=7 and remains constant for
Pr=50. The minimum value of Nusselt number
occurs at Pr=0.03. For W/D=2.5, figure (10), the
minimum value of Nusselt number obtains at
Pr=0.03, then the Nu increases by 2.5 at Pr=0.7.
The Nusselt number slightly increases at Pr=7
and its value do not change for Pr=50. For
W/D=5, figure (11), the Nusselt number
increases with increasing the Prandtl number.
The minimum Nusselt number value obtains at
Pr=0.03, then the Nusselt number increases by a
value of 2 at Pr=0.7. The Nusselt number
increases slightly to reach maximum value at
Pr=50.
16
Ra=10000
Ra=100000
Ra=100000
14
12
Nu
10
8
6
4
2
0
0.01
0.1
1
10
100
Pr
Figure (9) Effect of Prandtl number on the average Nusselt number for W/D=1.67.
12
Ra=10000
Ra=100000
Ra=1000000
10
Nu
8
6
4
2
0
0.01
0.1
1
Pr
10
100
10
Ra=10000
Ra=100000
8
Ra=1000000
Nu
6
4
2
0
0.01
0.1
1
10
100
Pr
372
CONCLUSIONS
Effect of Prandtl number on the natural
convection heat transfer from circular horizontal
cylinder in a square enclosure was investigated
numerically over a fairly wide range of Ra with
taking the effect of enclosure width. The main
conclusions of the present work can be
summarized as follows:
1. The numerical results show that the Nusselt
number increases with increasing the Raylieh
number for all cases.
2. The flow patterns and isotherms display the
effect of Ra, enclosure width, and Prandtl
number on the thermal and hydrodynamic
characteristics.
3. The results show that the streamlines and
those of other higher Prandtl numbers for all
enclosure widths and Ra≥105.
4. The Conduction is the dominant of the heat
transfer at Ra=104 for all Prandtl numbers. The
contribution of the convective heat transfer
increases with increasing the Raylieh number.
5. The streamlines and isotherms for Pr≥0.7 are
nearly similar and independent of Prandtl
number.
6. The results show that the Nusselt number for
Pr=0.03 is unique and differ from those of other
higher Prandtl numbers for all enclosure widths.
7. The enclosure width to the cylinder diameter
has an influence on the results of the Nusselt
number for Pr=0.03.
REFERENCES
Kuehn TH, Goldstein RJ., (1976). An experimental
and theoretical study of natural convection in
the annulus between horizontal concentric
cylinders. Journal of Fluid Mechanics, Vol.
74, pp. 695–719.
Ekundayo CO, Probert SD, Newborough M, 1998.
Heat transfer from a horizontal cylinder in a
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Moukalled F., Acharya S., (1996). Natural convection
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C. Shu; and Y. D. Zhu, (2002). Efficient computation
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Koca, H.F. Oztop, Y. Varol, (2007). The effects of
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(1985).
Numerical
Grid
Generation:
Foundations and Applications. Mississippi
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Roache, P., J., (1982). Computational Fluid
Dynamics. Hermosa publishers.
Ferziger J. H. and Peric M., (2002). Computational
Methods for Fluid Dynamics. Springer, New
York.
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374
USING MONTE CARLO SIMULATION TO CALCULATE THE DOSE
REDUCTION AT THE MAZE ENTRANCE OF A RADIOTHERAPY ROOM
Dindar Shamsadin Bari
Department of Physics, Faculty of Science, University of Zakho, Kurdistan Region – Iraq.
Abstract:
The aim of this study was to introduce a method to reduce the dose of the backscattered photons from
a linear accelerator at the maze entrance of a radiotherapy room (RR). For this purpose a typical RR was
designed and simulated using FLUKA Monte Carlo Code (version 2011.2b.1). The maze of a RR was the
main focus for the study. Its walls including, floor and ceiling were lined with thin sheets of lead and
stainless steel of 2 mm and 4 mm thicknesses respectively to find the most effective material and thickness
for dose reducing.
It was found that 2 mm lead sheet was able to reduce the dose at the maze entrance by more than 60 %
and 4 mm 70 %. Whereas, 2 mm of stainless steel was able to reduce about 30% of the dose and 4 mm was
able to reduce about 35%.
Keywords: Scattered photons, radiotherapy room, calculation of the dose at the maze, radiation protection,
FLUKA Monte Carlo Code.
1- Introduction:
adiotherapy is one of the main treatment
modality for cancer disease and external
beam radiation treatment is the most common
form of radiotherapy. Cancerous cells are
destroyed through damage caused by X-rays
ionising radiation. Energy is transferred to tissue
by the photon beam through particle interactions
within the tissue (Marcu et al, 2012).
Unfortunately, as we all should know, radiation
is a double-edged sword. Radiation that can treat
tumors and save lives can also cause cancer,
cataracts, etc. and is potentially lethal (Hall,
2005). Therefore, patients receiving radiotherapy
as well as staff working near linear accelerator
need to be shielded from high-energy radiation
doses. These two groups are protected in quiet
different ways: normally patients are protected
by direct shielding on the linear accelerator;
whilst staff and passerby are protected by mazes
and thick concrete walls.
The primary objective of designing a
radiotherapy room (RR) and its access maze
together called “bunker” is to ensure that dose
rate limits for staff and the public are not
exceeded and are kept as low as reasonably
practicable.
This
is
accomplished
by
constructing the walls of bunkers thick enough
from medium and high density materials such as
concrete, lead and steel to provide adequate
shielding. The degree to which photon beam is
attenuated depends upon the photon energy, the
atomic number and density of the elements in
R
375
the shielding material, the thickness of the
shielding (Amin, 2014).
The maze length and shape must be such that
there are satisfactory dose rates at the interface
with the outside RR. At this point there are two
factors contributing to the dose rate. The first
factor is the x-rays (photons) that are generated
from the interactions of primary beam within
patients, collimators, surface of walls of RR and
air in the room. It is found that the energy
spectra of those photons were not to exceed 400
keV with an average of about 100 keV (Biggs,
1991; Al-Affan et al, 1998; Al-Affan, 2000).
The second factor is the leakage radiation from
the treatment head. Leakage radiation penetrates
machine head and travels through the wall
adjacent to the maze entrance (Ionisation
Regulation Radiation, 1991).
Previously several actions were taken to
reduce the dose at the maze entrance such as
extending the maze length long enough or add a
turn with a small length in the maze (called legs)
in a different direction (Carinou et al, 1999).
However, extending the maze length whether in
one direction or adding another leg in a different
direction would certainly add to the cost of RRs,
occupy more space which can be a problem if
the situation was to upgrade an existing RR
within an area of a very limited space. A long
maze would also mean more time being taking
patients in and out of RRs; accordingly, it is
significant to determine another method to
reduce the dose.
Both lead and steel are widely used for
radiation protection purposes, because of their
high absorption of radiation. Lead has a very
high density and is a very good X- and gamma
ray shielding material, however, it is toxic and
more expensive than concrete (Xu, 2008). Steel
is also expensive compared to concrete, but is
not toxic (James, 2005). It is more efficient than
concrete as an X- and gamma rays shielding
material, but less efficient than lead. However, it
should be noted that steel has the advantage of
lower
photoneutron
production
(Biggs,
2010).The aim of this research was to focus on
the scattered photons which may contribute
more than 50% of the dose at the maze entrance
(Al-Affan and Smith, 1996; Al-Affan, 2000) and
how they can be reduced. This can be achieved
by lining the surface of the maze walls by
different metals (lead and stainless steel) with
various thicknesses by using a computational
method termed Monte Carlo simulation.
Leakage radiation could be treated in the future
study. The treatment head of a linear accelerator
can be modeled to produce leakage radiation and
see the level of leakage radiation at the maze
entrance and how it can be reduced either
through increasing the thickness of the wall
adjacent to the maze entrance or line the wall
surface with extra high density material such as
lead.
2- Method of Calculations:
This research presents a method using Monte
Carlo simulation to predict energy spectrum
based upon the ratio of photon backscattered
from the walls of the RR, particularly the maze
walls. The simulation was divided into three
different parts; in the first simulation, RR with
its access maze was modeled without any
change; in the second simulation, the walls of
the maze including its roof and floor were lined
with 2 and 4 mm lead sheet of density 11.25
g.cm-3 respectively; and in the third simulation,
walls of the maze including its roof and floor
were lined with 2 and 4 mm stainless sheet of
density 8.0 g.cm-3 respectively. The geometry of
the RR with its access maze is shown in Figure
1.
Figure 1: Diagram of a typical radiotherapy room used to calculate the dose for multi-scattered photons at the
maze entrance.
376
The room walls, roof and floor are made of
concrete of density 2.35 g.cm-3. Also, in this
simulation, it was assumed and suggested by
National Council on Radiation Protection &
Measurements (NCRP, 2005) that the concrete
had an elemental composition of (in percentage
by weight composition) 0.92% hydrogen,
49.83% oxygen, 1.71% sodium, 4.56%
aluminum, 31.58% silicon, 1.92% potassium,
8.26% calcium and 1.22% iron. The typical RR
including the maze was 1200.0 cm in length,
800.0 cm in width, and 300.0 cm height. All
walls had 100 cm thickness, and both floor and
roof thicknesses were 30 cm (that assumption
was used to speed up the computation without
compromising the results). The photon source
was fixed at 100 cm away from the surface of
the rectangular parallelepiped water phantom
that had a symmetric size of 50 cm × 50 cm × 50
cm a long beam axis. Moreover, in this
simulation the worst case of the beam was
selected (i.e. the highest expected dose at the
maze entrance). Although photons source from
the linear accelerator is divided into primary
photons and leakage photons, in the present
work, beam was assumed to have only primary
photons and leakage photons was ignored
because less amount of the dose at the maze
entrance comes from leakage photons in
compare to scattered photons. The photon beam
had a radius of 5.65 cm (at 100 cm from the
phantom surface) at the surface of water
phantom giving an equivalent area (field size) of
10 x 10 cm2.
In order to calculate doses of backscattered
photons and increase the efficiency of the
detector, a large rectangular parallelepiped water
detector was positioned at the entrance of the
maze and covered the maze as a door. This
377
detector approximately was 200.0 cm in length,
1 cm in depth, and 300 cm in height. This size
was restricted by the width and height of the
maze.
The whole geometry was surrounded by a
large sphere of Void of 1000 cm in radius and of
air medium, and this was surrounded by a larger
sphere of Black Hole of 100000 cm in radius.
The irradiations were carried out for a range of
photon energies (0.5, 1, 2, 3, 5, and 6 MeV) to
study several components of the X-ray spectrum
which are usually present in the primary beam
(of energies up to 10 MV). These energies were
taken because if the photon energy is greater
than (6-7 MeV or 10 MV) neutrons are
generated by photonuclear reaction in the
treatment head of the linear accelerator (Ongaro
et al, 1999). Therefore different actions need to
be taken for the purpose of dose reduction due to
scattered neutrons. For each energy value
FLUKA MC was run for 3 cycles to reduce
statistical fluctuation in the results; moreover, 1x
107 photon histories were followed for each
simulation to get the required statistical
uncertainty of better than 10%. Therefore,
calculations of the doses were taken for a long
time, which was about 100 hours per 3 cycles for
all statuses.
3- Results and Discussion:
Table 1 shows that the calculations made by
the detector that was placed at the entrance of
the maze. It can be seen that the ratio of the dose
at the maze entrance as shown in Fig. 1 when 2
mm lead was used to that with no lead (only
concrete wall) varies from 26% to about 39%
depending on the energy of the primary beam
generated by the linear accelerator.
Table 1: Dose with 2 mm lead (DL) and with concrete (DC) and their ratios at the entrance of the maze
DL
Energy (MeV)
(MeV/cm3) per
DC
(MeV/cm3) per
Percentage of
Ratio=( DL / DC)x100
statistical
%
particle (×10-7)
particle (×10-7)
0.5
0.82
3.16
26.0
8
1
1.1
3.93
28.0
9
2
1.85
5.56
33.26
5
3
1.96
5.43
36.12
7
4
1.95
5.67
34.41
8
5
2.08
5.59
37.27
6
6
2.25
5.77
38.90
9
uncertainty
However, in the Table 2 it can be seen that the ratio of the dose at the maze entrance when 4 mm
lead was used to that with no lead (only concrete wall) varies from 23% to about 28% depending on
the energy of the primary beam generated by the linear accelerator. Therefore it would be useful to
know the spectrum of the primary beam to be able to simulate the dose calculations at the maze
entrance. This would be interesting to show that 2 mm lead lining the maze would reduce the dose
scattered through the maze by more than 60% and 4 mm lead lining the maze would reduce more than
70% of the scattered dose.
Table 2: Dose with 4 mm lead (DL) and with concrete (DC) and their ratios at the entrance of the maze
Energy (MeV)
DL
DC
Ratio=( DL / DC)x100
Percentage of
(MeV/cm3) per
(MeV/cm3) per
%
statistical
particle (×10-7)
particle (×10-7)
0.5
0.74
3.16
23.0
8
1
1.2
3.93
26.0
9
2
1.66
5.56
30.00
5
3
1.40
5.43
26.00
7
4
1.49
5.67
26.27
8
5
1.86
5.59
33.24
5
6
1.60
5.77
28.00
7
uncertainty
378
Table 3 shows calculations made by same detector and under same conditions, only lining metal
was changed from lead to stainless steel. It can be noted that the ratio of the dose at the maze entrance
varies from 49% to 77% when 2 mm of stainless steel was used to that with no stainless steel .So, this
means that 2mm of stainless steel can reduce lesser amount of the scattered dose than 2 mm of lead.
This due to fact that stainless steel has lesser density than lead.
Table 3: Dose with 2 mm stainless steel (Ds) and with concrete (Dc) and their ratios at the entrance of the maze
Energy (MeV)
DS
DC
Ratio=( DS / DC)x100
Percentage of
(MeV/cm3) per
(MeV/cm3) per
%
statistical
particle (×10-7)
particle (×10-7)
0.5
1.57
3.16
49.84
8
1
2.82
3.93
71.95
6
2
4.43
5.56
79.67
7
3
3.97
5.43
73.16
7
4
3.64
5.67
64.23
8
5
3.92
5.59
70.03
6
6
4.49
5.77
77.80
4
uncertainty
However, in the Table 4 it can be seen that the ratio of the dose at the maze entrance when 4 mm
stainless steel was used to that with no stainless steel varies from about 50% to about 80% depending
on the energy of the primary beam generated by the linear accelerator. By comparing the calculations
of the Table 3 and Table 4, it can be noted that there was not much difference between the ratios of the
dose with 2 mm and 4 mm stainless steel sheets.
Table 4: Dose with 4 mm stainless (Ds) and with concrete (Dc) and their ratios at the entrance of the maze
DS (MeV/cm3)
DC (MeV/cm3)
Percentage of
Ratio=( DS / DC)x100
Energy (MeV)
379
per particle
per particle
statistical
%
(×10-7)
(×10-7)
0.5
1.57
3.16
49.84
8
1
2.55
3.93
65.04
7
2
3.93
5.56
70.68
10
3
3.54
5.43
65.24
9
4
3.28
5.67
57.84
6
5
3.62
5.59
64.79
8
6
4.63
5.77
80.24
9
uncertainty
In this study, it is clear that from the
calculations shown in Tables (1, 2, 3 and 4), 2
mm and 4 mm lead sheets could reduce more
scattered dose than stainless steel, and therefore,
it is better than stainless steel for the purpose of
the maze lining. In addition, 2 mm lead should
be the thickness of choice for the purpose of
maze lining and dose reduction at the maze
entrance, due to some Physical phenomenon
such as Photoelectric effect, Compton scattering
and Pair production which occurs at photon
energy more than 1 MeV and may contribute
with the scattered photons and after that increase
the dose.
Practical measurement of the dose from the
scattered photons at the maze entrance would be
harder than theoretically (computing) due to
contributions of the leakage radiation which may
effects the actual calculations. Leakage radiation
arises from the treatment of the linear
accelerator. Therefore, the treatment head must
be shielded to reduce it as low as practically
applicable. Otherwise there will be disagreement
between practical and computed calculations.
4- Conclusion:
The aim of the present research was to use
and compare two different shielding materials
for the purpose of dose reduction at the maze
entrance of radiotherapy rooms. After simulation
and design of RR with its maze, irradiation was
carried out for rang energies up to 6 MeV, it was
found that 2 mm lead sheet could reduce up to
about 60 % and 4 mm 70 % of the dose.
Whereas 2 mm of stainless could reduce about
30 % and 4 mm 35 %, of the dose of
backscattered photons at the maze entrance when
linear accelerator runs at the worst case (highest
expected dose at the maze entrance).
In this research, it was determined that lead
is the material of choice for the maze lining to
reduce the dose, because of its high photons
absorption compare to the stainless steel. In
addition, photons energy more than 10 MeV or
higher energies will lead to photoneutron
production and so another method needs to be
used to solve the problem. Therefore energies
lower than 10 MeV were selected.
5- References:
Al-Affan, I.M. and Smith C.W. (1996). Radiation
quality of scattered photons at the maze
entrance of radiotherapy rooms for photon
beams of energy 0.5-30 MeV. Radiation
Protection Dosimetry, 67, 299-302.
Al-Affan, I.M., Smith, C.W., Morgan, N.H. and
Lillicrap, S.C. (1998). Dose rate and energy
distributions of X rays from a linear
accelerator at maze entrance of a radiotherapy
room by measurement and Monte Carlo
simulation. Radiation Protection Dosimetry,
78, 273-277.
Al-Affan, I.M. (2000). Estimation of the dose at the
maze entrance for X-rays from radiotherapy
linear accelerators. Medical Physics, 27, 231238.
Amin, M. (2014). Properties of a Some (Ag-Cu-Sn)
Alloys for Shielding Against Gamma Rays.
International Journal of Advanced Science
and Technology, 63, 35-46.
Biggs, P,J. (1991). Calculation of Shielding Door
Thicknesses for Radiation Therapy Facilities
using the ITS Monte Carlo Program. Health
Physics, 61, 465–472.
Biggs, P.J. (2010). Radiation shielding for
megavoltage photon therapy machines.
Boston: Massachusetts General Hospital and
Harvard Medical School.
Carinou, E., Kamenopoulou, V. and Stamatelatos,
I.E. (1999). Evaluation of neutron dose in the
maze of medical electron accelerators.
Medical Physics, 26, 2520−2525.
Ferrari, A., Sala, P.R., Fasso, A. and Ranft, J. (2011).
Fluka: a multi-particle transport code [Online].
Available
at:
http://www.fluka.org/content/manuals/FM.pdf
(Accessed: 2 February 2015).
Hall, E. (2005). Radiobiology for the Radiologist (6th
ed.). Philadelphia: Lippincott Williams and
Wilkins.
Ionisation Regulation Radiation (1991). Health and
safety. London: The Stationery Office
Limited.
James, A.D., James, E.R., Raymond, K.W., Peter,
J.B., Patton, H.M., Richard, C.M. et al. (2005).
Structural shielding and evaluation for
megavoltage X- and gamma-ray radiotherapy
facilities. Washington: NCRP.
Marcu, L., Allen, B. and Bezak, E. (2012).
Biomedical physics in radiotherapy for cancer.
Collingwood: CSIRO Publishing.
Ongaro, C., Rodenas, J., Leon, A., Perez, J., Zanini,
A. and Burn, K. (1999). Monte carlo
simulation and experimental evaluation of
photoneuron spectra produced in medical
linear accelerators. Proceedings of the 1999
Particle Accelerator Conference (pp. 2531 2533). New York, 27 March -02 April.
Xu, S. (2008) .A Novel Ultra-light Structure for
Radiation Shielding. Master thesis, Carolina:
North Carolina State University.
380
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381
BULK ETCH RATE MEASUREMENT OF CR-39 DETECTOR AT
CONSTANT NORMALITY USING THE DIAMETERS AND THE LENGTHS
OF THE TRACKS OF α-PARTICLES
Hussein A. Ahmed
Dept. of Physics, Faculty of Science, University of Zakho, Kurdistan Region – Iraq.
Abstract
The aim of this paper is to determine the bulk etch rate of Nuclear Track Detector (type CR-39) using
the track’s diameter-length (Le-D) measurement method by irradiating the detector with different
energies of alpha particles from 241Am source. Constant normality (6.25) N of the chemical solution NaoH
at a temperature of (70±1) oC is used in etching the detectors to measure the track’s lengths and it’s
diameter at different etching times corresponding to the energy of the incident’s particles. The bulk etch
rate VB values of CR-39 detector have been found by using Le-D method are (1.4228, 1.2563, 1.5098,
1.438) µm.hr-1 and they showed good agreement with the values measured by the removed layer method
which were (0.944-1.831) µm.hr-1.
Key words: Bulk etch rate. CR-39, Track length, Nuclear Track Detector (NTD).
Introduction
A
heavy charged particle leads to
intensive ionization when it passes
through matter. Along the path of the particle, a
zone called the latent track is created, which is
enriched with free chemical radicals and other
chemical species. If a piece of material
containing the latent track is exposed to some
chemically aggressive solution (such as aqueous
NaOH or KOH solution) the chemical reaction
would be more intensive in the latent track. Such
a solution is called the etchant. Through the
etching, the latent track becomes visible as a
particle “track” which may be seen under an
optical microscope. The effect itself has been
known for long time, which is called the “track
effect” (Nikezic and Yu, 2006), The technique
has been extensively investigated in the
literature, and has been widely applied in many
fields of science and technology (Nikezic and
Yu, 2003). The main use of CR-39 plastic
detector is in the field of health physics, such as
for detection of proton, helium and heavy
charged particle, radon monitoring and neutron
dosimetry (Jain, et al., 2013)
Two main parameters that govern the
track formation are the bulk etch rate VB and the
track etch rate Vt. These two important variables
were introduced by Fleischer et al. (Fleischer et
al. 1975). The bulk etch rate VB is the rate of
removing of the undamaged surface of the
detector. Due to the chemical reaction between
the etching solution (etchant) and the detector
material, some molecules of the detectors are
removed. The final track is the removal of the
material from the detector surface.
During etching, the material is removed,
layer by layer, and the thickness of the detector
becomes smaller and smaller.
The bulk etch rate has been the subject of
many measurements in the past, and it is
probably the most frequently measured quantity
concerning track detectors. It has been
determined in a variety of materials, irradiated
by different ions and etched under various
etching conditions. There are several VB,
determination methods broadly categorized into
indirect and direct methods.
One of these methods is the determination
of VB from the diameter of tracks of fission
fragments; this is one of the oldest and most
frequently used methods for determination of
VB. Fission fragments are easily obtained from a
252
Cf radioactive source. Since the ranges of
fission products are relatively small, the track
etch rates can be considered constant during
etching. Under this condition, the following
simple approach can be applied. When the
incidence is normal and Vt = constant, the
diameter of the track opening is given by
(Nikezic and Yu, 2004):
=
(1)
where V = (Vt/VB), and h is the thickness of
the removed layer during etching.
The bulk etch rate could be determined
from the change in the mass of the detector ∆m
382
and the density of the detector material, VB
could be calculated as (Durrani and bull, 1987)
and it is based on Eq. (2):
∆
=
(2)
where Δm is mass difference, A the etched
surface area, ρ the density of the detector and t is
the etching time.
Nikezic and Janicijevic proposed a “peeloff” method to directly measure the bulk etch
rate for the LR-115 detector based on surface
profile measurements using an instrument called
(Taylor Hobson, Leicester, England).(Nikezic
and Janicijevic, 2002).
From the other methods of measurement
VB the track of high gamma dose on the
response of PADC detector (CR-39). The bulk
etch rate VB, alpha track etch rate Vt and the
sensitivity or etch rate ratio-V = Vt/VB were
measured based on measurements of the track
diameters for normally incident fission
fragments and alpha particles according to the
equation(Salman, 2010):
=
(3)
Where Df is the diameter of tracks resulting
from the fission fragments.
Also VB could be measured by another
method; and it is by measuring the cone base
diameter D, and the height Le, this method
requires selecting only tracks for which precise
measurements of the cone height and diameter
could be performed (for example we cannot
measure the track cone heights for low energies,
for which the microscope image may be affected
by shadow tracks).(Manzoor, 2006; Balestra et.
al., 2007).
=
[ +
+
]
(4)
Since VB is one of the most important
parameters that control the formation and
development of tracks, there has been a large
literature debate discussing VB. Here, an
incomplete survey of works on VB was given. It
has been shown that VB depends on many factors
like the purity of the basic substances, the
molecular structures of polymers, conditions of
polymerization, environmental conditions during
the irradiation and finally the etching conditions.
383
In this way, there are different topics related to
VB, which included the following:
_ Dependence of VB on the chemical
composition and preparation of the detector;
_ Dependence of VB on the etching
conditions (temperature and concentration of
etching solution as well as the presence of
stirring);
_ Dependence of VB on the irradiation of the
detector before etching with different kinds of
ionizing or non-ionizing radiations(Nikezic and
Yu, 2004).
Experiment
The CR-39 detectors used in the present
work were irradiated with alpha particles with
energies (2.0, 2.9, 3.6 and 4.3) MeV. An 241Am
alpha-particle source (main initial energy Eo
=5.48 MeV) was employed, and the stopping
medium between the source and the detectors
was air. The irradiated detectors were etched in
an aqueous 6.25 N NaOH solution at a
temperature of 70 ◦C for different duration times
of etching process. The lengths and the
diameters of the tracks were measured by using
a digital camera of type MDCE-5A fixed
directly on the light microscope and connected
to the computer by using Image Driving Software
which is provided with the camera for picturing
the lengths and the diameters of the tracks.
Results and discussion
There are two methods to find the bulk
etch rate for the used detector; it can be found
either by irradiating it with charged particles or
without irradiating it with those particles in the
methods of measuring the VB. There is a modern
method to measure VB, by measuring the length
- diameter of the track. This method (Le-D)
requires irradiating the detector with charged
particles and photographing the form of etching
tracks that were formed in the detector due to the
etching solution, and measure the length and the
diameter experimentally. While it requires the
other known methods - including the method of
measuring the thickness of the removed layer
from the surface of the detector - to measure the
thickness of the detector after etching operations
for successive periods of time without the need
to irradiate it with charged particles.
Figure (1 ) shows the relationship between
multiplying etching period in along the formed
track and the square of the track diameter (t*Le
& D2) at different energies of alpha particles.
Figure (2) shows the relationship between the
square of the track diameter and the square of
the track length (D2 & Le2) through different
periods of time with energies of alpha particles
(α) ( 2.0 , 2.9, 3.6, 4.3) MeV. It can be observed
from the figures that the relationship between
(t*Le & D2) and (D2 & Le2) are almost a linear
relationships in stage of cone regular track , and
that the relationship does not continue to change
linearly with the increasing of etching hours over
this stage , because the length of the track
increases through etching time and up to
permanence or saturation at the end of the first
stage when it reaches the top of the etching track
to end over the particle in the detector , while the
diameter of the track continues to increasing
with the progress of etching at the stage of overetching that extent outside the range of the
particle in the detector. Thus, our measurements
must be within the regular phase of the cone
shape of the track, where we have performed
measurements for different times of etching.
The stoppage of etching at the times that
shown in the figure 1 and figure 2 is attributable
to the lengths of the formed tracks do not
continue to increase with the increasing of the
tracks diameters with the progress of etching; R'
should not have a negative value when etching
solution reaches a depth greater than the extent
of the particle inside the detector and after overetched of the detector.
To measure VB in a method of the
removed layer thickness from the detector by
etching
without irradiating it with alpha
particles (by etching the detector in a
background ), just by etching CR-39 detector by
using the etchant solution itself NaOH at
temperature (70 ± 1) oC at the same normality.
Figure 3 shows the relationship between
the bulk etch rate( VB) using the method of Le-D
and energies of alpha particles were used to
irradiate the detector CR-39. It could be seen
from the figure that the bulk etch rate VB is
independent on energies of α-particles (i.e. the
relation is constant). The values of VB are
around 1.4 µm /hr.
Fig. (1): The relationship between the square of the track diameter (D2) and the product of etching time (t) and
height (Le) at different energies of alpha particles.
384
Fig. (2): The relationship between the square of the track diameter (D2) and square of height (Le) at different
energies of alpha particles.
Fig. (3): The relationship between the bulk etch rate (VB) and the energies of alpha particles at constant
normality.
385
This value is rather consistent with the value of VB obtained by the method of Le-D and the method of
measuring the thickness of the removed layer as shown in Figure 4 which has been proved by (AlNia'emi and Kasim., 2013) and the value of VB is 1.23 µm /hr (Ho et al., 2003) and the value of VB is
1.45 µm /hr (Ahmed, 2010) and the value of VB is 1.7 µm /hr (Yu et al., 2005) by using NaOH at the
same normality (6.25 N).
Fig. (4): Rates of the bulk etch using the method of the removed layer thickness and the method Le-D with
different normality (Al-Nia'emi and Kasim, 2013).
Conclusions
References
From the obtained results we conclude that
the method Le-D gives good results to measure
the bulk etch rate (VB), but it's not a simple
method because it requires to get photos of the
tracks in the detector, measurement of the
lengths and the diameters accurately. Also the
bulk etch rate does not depend on energy of the
particles that is used to irradiate the detector. It
was found that the results of VB that were
measured by using the thickness of the removed
layer are rather compatible with the results of VB
that were measured by using Le-D method that
requires irradiating the detector with the particle
charged with different energies.
Ahmed, H.A., (2010). M.Sc. Thesis, Physics
Department, College of Education,
University of Mosul, Iraq.
Al-Nia'emi, S. H. S., and Kasim., Y. Y. (2013),
“Determination of the Bulk Etch Rate of
the Nuclear Track Detector CR-39 Using
Le-D Method”, ‫ المجلة االردنية للفيزياء‬Vol. 6,
PP. 17-25.
Balestra, S., Cozzi, M., Giacomelli, G.,
Giacomelli, R., Giorgini, M., Kumar, A.,
Mandrioli, G., Manzoor, S., Margiotta,
A.R., Medinaceli, E., Partizii, L., Popa,
V., Qureshi, I.E., Rana, M.A., Sirri, G.,
Spurio, M., Togo, V. and Valieri, C.,
(2007), "Bulk etch rate measurements and
386
calibrations of plastic nuclear track
detectors" Nucl. Instr. and Meth., in:
Physics Research, B 254 254.
Durrani, S.A., and Bull, R.K., (1987), "Solid
State
Nuclear
Track
Detection",
(Pergamon Press, Oxford).
Fleischer, R.L., Price, P.B., and Walker. ,R.M.
(1975). "Nuclear Tracks in Solids",
University of California Press, Berkley.
Ho, J.P.Y., Yip, C.W.Y., Nikezic, D., and Yu,
K.N. (2003), "Effects of stirring on the
bulk etch rate of CR-39 detector" Radiat.
Meas., R 36, PP. 141-146.
Jain, R. K., Ashok Kumar, Chakraborty, R. N.
and Nayak , B. K. (2013), “The Response
of CR-39
Plastic Track Detector to
Fission
Fragments
at
Different
Environmental (Temperature) Conditions”
Nucl. Phys., Vol. 58, PP 564-565.
Manzoor, S., (2006)."Nuclear Track Detectors
for Environmental Studies and Radiation
Monitoring",
(Physical
Department,
University of Bologna,
Nikezic, D., and Janicijevic, A. (2002), "Bulk
etching rate of LR-115 detectors", Appl.
Radiant. Isot. 57, 275-278.
387
Nikezic, D., and Yu, K.N. (2003), "ThreeDimensional Analytical Determination of
the Track Parameters: Over-Etched
Tracks", Radiat. Meas.,Vol.37, PP. 39-45.
Nikezic, D., and Yu, K.N. (2004), "Formation
and Growth of Tracks in nuclear Track
Materials", Materials Science and
Engineers R 46, PP. 51-123.
Nikezic, D., and Yu, K.N. (2006), "Computer
Program Track-Test for Calculating
Parameters and Plotting Profiles for Etch
Pits in Nuclear Track Materials",
Computer Physics Communications, Vol.
174, PP. 160-165.
Salman, T.M. (2010), "The effect of stirring and
interruption on the etching characteristic
of cr-39 track detector", Journal of Basrah
Researches ((Sciences))Vol. 36, PP 18172695.
Yu, K.N., Ng, F.M.F. and Nikezic, D. (2005),
"Measuring depths ofsub-micron tracks in
a CR-39 detector from replicas using
Atomic Force Microscopy", Radiation
Measurements Vol. 40 PP. 380 – 383.
‫‪Journal of University of Zakho, Vol. 2(A) , No.2, Pp 382-388, 2014‬‬
‫پێوانهيى بارستاييى ڕێژە ھهڵدەكۆڵى ‪ CR-39‬دۆزەرەوە له نۆمالهتيى نهگۆڕ لهاليهن بهكار ھێنانى تيرەى بازنهيهكه و‬
‫درێژيهكهى ڕێيهكه‬
‫كورتي‬
‫ئامانجێ ئهڤ كاخهزە دا ڕێژە ێێ بارستايى ھهڵدەكۆڵى دۆزەرەوەێ ڕێ ناووكى ديارى بكه )جوور ‪ ( CR-39‬تيرەى‬
‫بازنه‪-‬درێژى ێ ڕێ بهكار دھێنه )‪ (Le-D‬ڕێگهێ پێوانه ب يررادياتينگێ دۆزەرەوە ب وزەى جياوازێ تهنۆچكهێ‬
‫ئهلفا ددە ‪ 241Am‬ژێدەر ب نۆمالهتيى نهگۆڕ ‪ (6.25)N‬ئێنێ ناۆه ێ ل كيمياوى چارەسهرى ‪ NaOH‬پلهى گهرميهكێ‬
‫‪ (70±1) oC‬ئۆك ھات بهكار ھێنان د ناڤ ھهڵدەكۆڵيێ دۆزەرەوە دا درێژى ێ ڕێ بپێوە ئوو وى تيرەى بازنه ل‬
‫ھهڵدەكۆڵيى جياواز حهيام نامه دگۆڕنهوە بۆ وزەيێ تهنۆچكه ێ ڕووداو ‪.‬بهھا ێێ ‪ CR-39‬بارستاييێ ڤب ھهڵدەكۆڵى‬
‫ڕێژە دۆزەرەوە بوويه دۆزييهەوە ب بهكار ھێنان ڕێگهێ –ديه ‪ (1.4228, 1.2563, 1.5098, 1.438) µm.hr-1‬ئوو‬
‫وان ڕێككهوتنى باش شان دا ب بهھا پێوا ب ڕێگهێ چين ال برد كۆ بوو‪. (0.944-1.831) µm.hr-1‬‬
‫@@‬
‫@@‬
‫‪@@Šbqýa@ßaí aì@ŠbÔa@ãa‡ƒnbi@ónibq@óîŠbïÈ@‡åÈ@CR-39@Ò’bÙÝÜ@ãbÉÜa@Â“ÕÜa@ß‡Éà@lby‬‬
‫‚‪@@Zó–þ‬‬
‫‪@‹qýa@‹ÔM‬‬
‫‪@ ßí @‘bïÔ@óÕî‹ @ãa‡ƒnbi@HCR-39@ËíäI@ñìíåÜa@‹qýa@Ò’bÙÜ@ãbÉÜa@Â“ÕÜa@ß‡Éà@‡î‡znÜ@íè@szjÜa@a‰è@æà@Ó‡a‬‬
‫)‪@ð÷bïáïÙÜa@ ßíÝzáÝÜ@ (6.25N)@ ónibq@ óîŠbïÉi@ 241Am@ Š‡—¾a@ æà@ bÑÜa@ pbáï§@ óÑÝn¬@ pbÔbi@ Ò’bÙÜa@ ÊïÉ“m@ Öî‹ @ æÈ@ (Le-D‬‬
‫‪@óÔbÜ@ bÉjm@ óÑÝn¬@ Â“Ô@ paÐ@ ‡åÈ@ bèŠbÔaì@ Šbqýa@ ßaí a@ ‘bïÔ@ ‹ÍÜ@ Ò’aíÙÜa@ @ Â“ÕÜ@ (70±1) oC@ òŠa‹y@ óuŠ†@ ‡åÈ@ NaOH‬‬
‫‪(1.4228, @ðè@@Le-D@óÕî‹ @ãa‡ƒnbi@p‡uì@Üa@@CR-39@Ò’bÙÝÜ@VB@ãbÉÜa@Â“ÕÜa@ß‡Éà@âïÔ@oäbØì@NóÔbÜa@pbáï§a‬‬
‫‪@@ðè@ Üaì@ óÜa¾a@ óÕjÜa@ óÕî‹ @ ãa‡ƒnbi@ óÝ—zn¾a@ w÷bnåÜa@ Êà@ ‡ïu@ ÖÐaím@ p‹éÄaì@ 1.2563, 1.5098, 1.438) µm.hr-1‬‬
‫‪N@(0.944-1.831) µm.hr-1‬‬
‫‪388‬‬
‫طوظارا زانكؤيــا زاخــؤ‬
‫أ ‪ -‬زانست‬
‫ثةربةندا (اجمللد) ‪2‬‬
‫هذمارا (العدد) ‪2‬‬
‫كانونا ئَي َ‬
‫كى‬
‫كانون األول‬
‫‪2014‬‬
‫َ‬
‫عرياقي‬
‫َ‬
‫هةريا كوردستانا‬
‫وةزارةتا خويندنا باال‬
‫وتو َيذينةوةي زانسيت‬
‫زانكــؤيــا زاخــــؤ‬
‫اقليم كوردستان العراق‬
‫وزارة التعليم العالي‬
‫والبحث العلمي‬
‫جامعــة زاخـــــو‬
‫طــــوظـــــارا‬
‫زانكؤيــا زاخــؤ‬
‫أ ‪ -‬زانست‬
‫مـجــلـــة‬
‫جامعة زاخــو‬
‫أ ‪ -‬علوم‬
‫ثةربةندا (اجمللد) ‪2‬‬
‫هذمارا (العدد) ‪2‬‬
‫كانونا ئَي َ‬
‫كى‬
‫كانون األول‬
‫‪2014‬‬

Issue 2 - The University of Zakho

Transcription

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