Journal of Life Sciences 6: 1100-1108

Journal of Life Sciences

Volume 6, Number 10, October 2012 (Serial Number 54)

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JLS Journal of Life Sciences

Volume 6, Number 10, October 2012 (Serial Number 54)

Contents

Biochemical and Molecular Biology

1077 The Study of Cholesterol Content in Synbiotic Fermented Dairy Products

Ilze Beitane and Inga Ciprovica

1082 Metabolic and Endocrine Responses of Desert-Adapted Mice Reproductive System to Increased Salinity

Elena Bukovetzky, Fuad Fares and Abraham Haim

1094 Influence of Abiotic Elicitors on Accumulation of Thymol in Callus Cultures of Origanum vulgare L.

Abedaljasim M. Aljibouri, Ashwaq S. Abd, Duha M. Majeed and Eman N. Ismail

1100 Low Peptone Dose as Inductor of Alkaline Protease Promoter Used for Invertase Gene Expression in Yarrowia lipolytica

Łukasz Śnieżewski, Ewa Walczak, Zbigniew Lazar and Małgorzata Robak

Biomedicine

1109 In Vitro Study on Virulence Potentials of Burkholderia pseudomallei Isolated from Immunocompromised Patients

Hadeel Tawfiq Al-Hadithi Rana and Muhammad Abdulnabi

1117 The Control of Malaria among PLWHA in Calabar, Cross River State, Nigeria

Patience Edoho Samson-Akpan, Olaide Bamidele Edet, Ekaette Francis Asuquo, Mary Achi Mgbekem and Idang Neji Ojong

1124 Spatiotemporal Distribution of Phlebotomine Sand Flies (Diptera: Psychodidae) in a Focus of Cutaneous Leishmaniasis in Foum Jamâa (Azilal, Atlas of Morocco)

Hassan Arroub, Abdelaaziz Alaoui, Hicham El Miri, Meryem Lemrani and Khalid Habbari

1133 Determination of Fungal Colonization in the Oral Cavity of College Students

Floridia Ricardo, Rodriguez Graciela, Ampuero Verónica and Gonzalez Luis

1142 Preliminary Results of Crayfish Distribution and Diseases in Latvia

Inese Briede

Interdisciplinary Researches

1145 The Effects of Simultaneous Application of Different Organic and Biological Fertilizers on Quantitative and Qualitative Characteristics of Cucurbita pepo L.

Mohsen Jahan, Alireza Koocheki, Mohammad-Kazem Tahami, Mohammad-Behzad Amiri and Mahdi Nassiri-Mahallati

1150 The Impact of Deforestation in Anambra State: The Ekwusigo Example

Joel Ekwutosi Umeuduji and Chukwuma Onyebueke Egbuonu

1158 Contribution of Study Bioecology of the Fauna Chamaerops humilis in the Region of Tlemcen (Algeria)

Damerdji Amina

1167 The Floristic Diversity of the Tlemcen Southern Slope Scrublands (Western Algeria)

Belhacini Fatima and Bouazza Mohammed

1174 Tools for Protein Structure Prediction at the bri-shur.com Web Portal

Sergey Feranchuk, Ulyana Potapova, Vladimir Potapov, Dmitry Mukha, Vladimir Nikolaev and Sergei Belikov

1180 The Elaboration of Horse Meat Products Technology

Аbzhanova Sholpan, Kizatova Мaigul, Мukhtarkhanova Rauan, Тarakbaeva Raushan and Аbilmazhinova Nazum

Journal of Life Sciences 6 (2012) 1077-1081

The Study of Cholesterol Content in Synbiotic

Fermented Dairy Products

Ilze Beitane and Inga Ciprovica

Faculty of Food Technology, Latvia University of Agriculture, Jelgava LV-3001, Latvia

Received: April 11, 2012 / Accepted: June 22, 2012 / Published: October 30, 2012.

Abstract: The influence of prebiotics as lactulose as well inulin on the ability of Bifidobacterium lactis to reduce cholesterol in milk was studied during milk fermentation. Pasteurized milk, freeze-dried starter culture Bb-12 (Bifidobacterium lactis, Chr. Hansen, Denmark), inulin—RAFTILINE®HP (ORAFI, Belgium), syrup of lactulose (Duphalac®, the Netherlands) in following concentrations: 0, 1%, 2%, 3%, 4% and 5% were used for experiments. The fermentation process was realized at 37 °C for 16 h. The content of cholesterol was determined according to AOAC Official Method 976.26A. The results showed that it is possible considerable to lower the level of cholesterol in fermented milk using B. lactis. The ability of B. lactis to decrease the level of cholesterol in milk can be influenced with addition of prebiotics. The lower concentration of cholesterol was determined in fermented samples with 4% of lactulose (9.5 mg/100g) and with 1% of inulin (10.4 mg/100g). Evaluating the influence of prebiotics on cholesterol content in fermented milk samples, it is obvious that the influence depends on the type of prebiotics (P > 0.05) and their concentrations (P < 0.05). Key words: Cholesterol, B. lactis, lactulose, inulin, fermented milk.

1. Introduction

Elevated total cholesterol and LDL (low-density

lipoprotein) cholesterol levels are widely

represented as a contributory risk factor for the

development of artherosclerosis, coronary heart

disease and stroke [1-3]. It has been reported that

hypercholesterolemia promotes to 45% of heart

attacks in Western Europe and 35% in Central and

Eastern Europe [4]. In addition, it is known that high

cholesterol levels and mortality are close related [5].

Manson et al. [6] have pointed out that even a 1%

reduction in serum cholesterol could reduce the risk

of coronary heart disease by 2-3%. Therefore it is

important to control the cholesterol intake by food

and to use the products with the ability to lowering

the blood cholesterol level. Fermented milk products

Inga Ciprovica, Ph.D., Prof., research field: dairy science and technology. E-mail: [email protected].

Corresponding authors: Ilze Beitane, Ph.D., Assist. Prof., research field: functional dairy products. E-mail: [email protected].

have been recommended as dietary supplements

because of their hypocholesterolemic effect in

humans [7]. Evaluating the relationship between

LAB (lactic acid bacteria) and the serum cholesterol,

it has found that lactobacilli or bifidobacteria can

exhibit hypocholesterolemic properties in humans

[8-10]. The possible mechanism it could be, that

LAB with active bile salt hydrolase or products

containing them have been suggested to lower

cholesterol levels through interaction with host bile

salt metabolism [11]. Bifidobacteria are one of the

most important probiotics associated with human

health. They have varied positive influence on

human health, inter alia, the lowering of serum

cholesterol in blood [10]. Xiao et al. [10] observed

that consumption of Bifidobacterium milk leads to a

meaningful reduction in triglyceride, low-density

lipid and total cholesterol. Therefore it is important

to produce products with low cholesterol content or

products which compounds should be reduced

The Study of Cholesterol Content in Synbiotic Fermented Dairy Products

1078

cholesterol level in blood by regular intake. One of

the possibilities is to add probiotics and prebiotics in

dairy products. The numerous studies indicate that

bifidobacteria have the ability to assimilate

cholesterol [12-14] which should be promoted with

adding prebiotics. There are limited studies [12]

about the influence of prebiotics on the ability of

bifidobacteria to reduce the level of cholesterol in

fermented milk. Therefore the task of the research

was to investigate the influence of lactulose and

inulin on the ability of Bifidobacterium lactis to

reduce cholesterol level in fermented dairy product

during milk fermentation.

2. Materials and Methods

Pasteurized milk with fat content 2.5% and the

strain of Bifidobacterium lactis (Bb-12, Chr.Hansen,

Denmark) was used for experiments. During the

experiments, the culture was maintained at -18 °C. As

prebiotics were used inulin RAFTILINE®HP (ORAFI,

Belgium) with polymerization degree ≥ 5 and degree

of purity 99.5% and syrup of lactulose (Duphalac®,

the Netherlands) with following composition (%):

lactulose—no less than 67, lactose—less than 6,

galactose—less than 10.

Different lactulose and inulin concentrations (1%;

2%; 3%; 4% and 5%) were added individually to 100

g of milk. Bifidobacterium lactis was inoculated with

2 mL of milk suspension (106 cfu/mL) and cultured at

37 °C for 16 h. The control sample was prepared

without the prebiotics for comparing with the obtained

results.

The level of cholesterol was determined according

to AOAC Official Method 976.26A.

The differences at the level of cholesterol were

analyzed using the analysis of variance (ANOVA).

t-test was applied to compare the mean values, and

P-value at 0.05 was used to determine the significant

differences. Experiments were carried out in triplicate.

3. Results and Analysis

Cholesterol is included in the membrane of fat

globules, and it makes up to 95% of the total sterol

content [15], the others 5% are cholesteryl esters. The

cholesterol content in milk is within the range from

0.09 g/L to 0.22 g/L, on average 0.16 g/L [16]. The

cholesterol content in milk is possible to decrease by

different techniques as cholesterol distillation, complexes

with cyclodextrins as well microorganisms [13].

Consequently, ability of Bifidobacterium lactis to

decrease the cholesterol level in fermented milk, as

well as the content of decreased cholesterol level

influenced by added prebiotics was studied.

The level of cholesterol in milk, control and in

fermented milk samples with different concentrations

of lactulose and inulin is presented in Fig. 1.

Fig. 1 The level of cholesterol in milk, control and in fermented milk samples with different concentrations of lactulose and inulin.

18.0

16.0

14.0

12.0

10.0

8.0

6.0

4.0

2.0

0.0

16.0

12.1 10.9

10.410.9

10.511.5 11.2

9.510.6 10.4

10.9


1079

The research results showed that it is considerable

possible to lower the level of cholesterol in milk using

B. lactis. The ability of B. lactis to decrease the level

of cholesterol in milk can be induced with adding the

prebiotics. The lower content of cholesterol was

determined in fermented milk samples with 4% of

lactulose (9.5 mg/100g) and with 1% of inulin (10.4

mg/100g). It confirms the conclusions of Palframan et

al. [17] that a greater bifidogenic effect of inulin was

obtained in 1% concentration. It indicates on the

relationship between the multiply of B. lactis and the

assimilation of cholesterol in milk. In preliminary

studies were established the ability of inulin and

lactulose to promote the growth of B. lactis in milk

(Table 1).

The results showed that the highest amount of B.

lactis did not provide the lowest level of cholesterol in

fermented milk with prebiotics. It is linked with the

activity of cholesterol esterase to catalyze the

hydrolysis of cholesteryl esters [19], thereby it should

be decreased the level of cholesterol in fermented milk.

The beneficial influence of lactulose on cholesterol

level in fermented milk can be explained with the

ability of lactulose to promote the growth of

bifidobacteria comparing with other prebiotics [20, 21].

Evaluating the research data it should be concluded

that influence of different prebiotics is not significant

(P > 0.05). The type of prebiotics has not significant

influence on the content of cholesterol in milk. The

significant decrease was determined in fermented milk

samples with different concentration of prebiotics (P <

0.05). There is established between control sample

and fermented milk sample with 4% of lactulose.

Summarizing the research results it should be

induced that the considerable decrease of cholesterol

content should be reached up to 25% during milk

fermentation. This tendency is possible to facilitate by

using appropriate prebiotics.

4. Discussion

In scientific articles there are achievements that

consumption of fermented dairy products significantly

decreases the cholesterol level in blood serum [22, 23]

and bifidobacteria have the ability to lower serum

cholesterol level in humans [14]. Manning et al. [24]

have indicated the ability of lactic acid bacteria to

decrease the total and LDL cholesterol level in blood.

Whereas Kiessling et al. [25] in study with human

reported about increase of high-density lipoprotein

(HDL) level but no reduction in total cholesterol (P =

0.001) in subjects fed yoghurt with Lactobacillus

acidophilus and B. longum. Contradictory data in

literature show the necessity to continue the research

in this field, because the mechanism how it happens is

not quite clear yet. The production of bile salt

hydrolase has been suggested as one of possible

mechanisms [26].

The effect of lactic acid bacteria is inconsistent;

there is possible the significant decrease of cholesterol

content and also unchangeable cholesterol content. It

depends mainly on the bacteria species used for

fermentation [27]. Pereira’s [12] research has

confirmed that depending on the species of bacteria

the decrease of the cholesterol content is possible from

0.4% to 47% in the selective culture mediums. Zhao

et al. [28] reported that Lactobacillus acidophilus was

Table 1 The influence of the concentrations of lactulose and inulin on the growth rate of B. lactis in fermented milk samples, lg cfu·mL-1 [18].

Concentrations(%) Lactulose Inulin 1 8.5 b 8.6 b 2 8.8 b 8.5 b 3 8.6 b 8.0 b 4 8.3 a 9.3 b 5 8.9 b 8.3 a Control 8.3 a 8.3 a

a: no disparity (P > 0.05) compared to control; b: a disparity (P < 0.05) compared to control.


1080

effective in reducing cholesterol level in MRS

medium. Whereas Ziarno et al. [13] have indicated

that Lactobacillus acidophilus and Bifidobacterium

spp. in fermented milk are able to assimilate

cholesterol from 18% to 38%.

The results of the research have shown that it is

possible to achieve a considerable decrease of the

cholesterol content if B. lactis is used for milk

fermentation. The obtained results have confirmed

with the statements expressed in literature about the

ability of lactic acid bacteria and bifidobacteria to

decrease the cholesterol content in milk [29].

Consequently, it may be maintained, that B. lactis is

able to influence the cholesterol content in fermented

milk. It is obvious that the influence depends on the

type of the used prebiotics (P > 0.05) and their

concentrations (P < 0.05). A parallel may be drawn

with information described in literature. Delzenne et

al. [30] have indicated to ability of inulin to suppress

the synthesis of triglycerides, so decreasing the

cholesterol level in blood. Roberfroid [31] reported if

2.5% of fructo-oligosaccharides were added to yogurt

it was possible to facilitate the decrease of cholesterol

in blood. Similar tendencies were obtained in the

research where the cholesterol content in fermented

milk samples was considerable decreased by adding

lactulose (P < 0.05) and inulin (P > 0.05). In general

the research results confirm the achievements reported

in literature about the ability of bifidobacteria, in this

case of B. lactis, to assimilate the content of

cholesterol in milk.

5. Conclusions

The ability of B. lactis to decrease the level of

cholesterol in fermented milk should be induced with

adding the prebiotics. The lower content of cholesterol

was determined in fermented milk samples with 4% of

lactulose (9.5 mg·100g-1) and with 1% of inulin (10.4

mg·100g-1). It is obvious that the influence depends on

the type of the used prebiotics (P > 0.05) and their

concentrations (P < 0.05).

Acknowledgments

Publication and disamination of research results has

been made due to the funding of the ERAF Project

“Promotion of scientific activities of LLU”, Contract

Nr. 2010/0198/2DP/2.1.1.2.0/10/APIA/VIAA/020.

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103-148.


Metabolic and Endocrine Responses of Desert-Adapted

Mice Reproductive System to Increased Salinity

Elena Bukovetzky1, Fuad Fares1 and Abraham Haim1, 2

1. Department of Evolutionary and Environmental Biology, University of Haifa, Mount Carmel, Haifa 31905, Israel

2. The Israeli Center for Interdisciplinary Studies in Chronobiology, University of Haifa, Mount Carmel, Haifa 31905, Israel

Received: March 12, 2012 / Accepted: April 27, 2012 / Published: October 30, 2012.

Abstract: From an evolutionary point of view, reproduction timing is an important adaptation which enables the transfer of genetic properties, thus enabling species continuation. Rodents inhabiting arid environments need reliable cues for triggering their reproduction. Results of previous studies showed that increased dietary salinity plays an important role as an ultimate regulator for desert adapted rodents’ reproductive system. The authors aimed discovering pathways by which high salinity can affect the reproductive system and metabolic status of desert adapted common spiny mice, Acomys cahirinus. Mice were challenged with osmotic stress, water source salinity increased gradually from 0.9% - 5% NaCl under short days (SD) and long days (LD). The authors assessed leptin and free fatty acid (FFA) levels using ELISA while, SYBR green technology was used for relative receptor expression (RQ) of target genes. Results revealed that serum levels of the hormone leptin were significantly (P < 0.05) reduced in salinity treated (ST) mice. Levels of FFA were significantly (P < 0.05) increased in LD- and SD-ST-males. In ST-SD females a significant increase (P < 0.05) in expression levels of leptin (Ob-Rt) mRNA receptor gene, in ovaries was noted. Aldosteron (Nr3c2) and vasopressin (AVP) mRNA receptor expression genes levels were significantly (P < 0.05) increased in both LD- and SD- ST- males. Key words: Acomys cahirinus, salinity, desert-adapted, AVP, Nr3c2, Ob-Rt receptor genes, leptin, FFA.

1. Introduction

Mammals are known to use various environmental

cues to forecast the occurrence of the optimal season

for reproduction. One such cue most commonly used

by them is day length changes (photoperiod) [1, 2],

which is a common cue in predictable ecosystems

with minimal year to year changes in climatic

conditions. Desert regions are unpredictable and

therefore, additional cues for successful breeding are

needed. Because of unpredictable climatic changes,

desert species could be “misled” if they entirely rely

only on photoperiod. However, photoperiod is used as

an initial cue for breeding regulation of desert-adapted

rodents [3-5].

Corresponding author: Elena Bukovetzky, Ph.D. candidate,

research fields: environmental biology and endocrinology. E-mail: [email protected].

The past management of agricultural land and

climatic changes, alone or acting together, have led to

the accumulation of excessive salts in land, water and

vegetation—a phenomenon named salination. At

sufficient levels, salinity has negative impacts on

human and natural assets such as plants, animals,

aquatic ecosystems, infrastructure, water supplies and

agricultural land [6]. Salt known as sodium chloride

(NaCl) holds a unique position in the annals of human

and animal history and in health and disease research.

Both the level of dietary NaCl and the background

diet are important in generating a hypertensive

phenotype in the rat [7]. But in the last years,

increased salinity levels in plants attracted researchers

with regards to possible effects on reproduction,

especially in desert adapted rodents [4, 5, 8]. Due to

increased evaporation under desert conditions, particle

Metabolic and Endocrine Responses of Desert-Adapted Mice Reproductive System to Increased Salinity

1083

concentration in plants elevate as the dry season

progresses since the last water input into the

ecosystem. It was suggested that increased dietary

salinity could be used as an ultimate signal for

reproductive system regulation of the desert adapted

golden spiny mouse Acomys russatus [4]. The results

of previous studies showed a strong negative effect of

high salt diet on the reproductive status of

desert-adapted males and females of A. russatus but

not on a mesic population of common spiny mice

Acomys cahirinus [5]. Sheep females, kept on high

salt intake diet during pregnancy showed hormonal

changes, which can negatively affect the offspring via

a mechanism called fetal programming [6]. High

salinity gradually treated rodents showed a significant

decrease in body mass (Wb). It has previously been

suggested that high salt consumption decrease food

intake in sheep [9].

Another mechanism by which the increased dietary

salinity affects reproduction in rodents could be

associated with the hormones vasopressin [4, 5] and

the hormone leptin, the latter produced by adypocites,

which regulates Wb, metabolism and reproduction [10, 11].

Salinity treatment to desert adapted A. russatus,

revealed a significant decrease in Wb, progressive

reproductive hiatus (decreasing number of estrous

cycles and relative decreased testis mass), which was

coupled with a massive (about 80%) reduction of

white adipose tissue (WAT) mass, compared with

their control group [5].

The primary role of WAT commonly referred to as

“fat” in mammals [12], is to store free fatty acids

(FFA) as triglycerides (triacylglycerol) during periods

of caloric excess (lipogenesis) and to mobilize these

reserves and release free fatty acids for ATP synthesis

via the Kreb’s Cycle during periods when expenditure

of calories exceeds the intake (lipolysis) [12]. WAT is

now recognised as a multifunctional organ; in addition

to the central role of lipid storage, it has a major

endocrine function secreting several hormones,

notably leptin. Clearly, the emerging picture of WAT

as an endocrine and immunological organ incorporates

cross-talk and co-stimulation of a multitude of

hormonal signals [13]. It is possible that localized

hormonal positive and negative feedback loops

interact with the tissue itself and/or through additional

pathways.

By the identification of leptin, the

adipocyte-derived hormone, and the leptin gene (Lep

Ob), followed by its cognate receptor Ob-Rt located

within the ventral medial hypothalamus (VMH),

paved the way for this area of research. Leptin is a 167

amino acid residue hormone almost exclusively

derived from WAT that shares structural similarities

with cytokines. The primary effects of leptin involve

the mobilization of lipolytic pathways of energy

expenditure as it signals to the hypothalamus that

energy sufficiency has been met [14].

In rodents it is well established that leptin gene

expression and plasma levels are proportional to total

body WAT stores [15]. Recently, it has been reported

that leptin has a role in the early onset of

reproduction [16, 17]. In mice it was noted that leptin

serves as a permissive signal for the reproductive

system [18]. Results of studies on bats have shown

that serum leptin levels are positively correlated with

WAT mass and negatively correlated with the

reproductive function [19, 20]. Leptin has been shown

to promote sexual maturation in various rodent species

and its role in reproduction has been investigated at

various sites within the hypothalamo-pituitary-gonadal

axis [21]. It was suggested that the pituitary-derived

leptin acts on the gonads and many other organs

equipped with leptin receptor through endocrine

function in addition to paracrine action with pituitary

hormone secretory cells [22].

By using a transgenic animal model, it was shown

that brown adipose tissue (BAT) expresses

mineralocorticoid receptor (MR), which can be

activated by aldosterone [23]. Little is known about

the potential involvement of the mineralocorticoid

system in WAT development. The results of previous


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pharmacological studies have suggested that

aldosterone may promote adipogenesis [24, 25]. It was

noted that chronic exposure to aldosterone induces

morphological, biochemical and molecular markers of

adipose conversion by stimulating the adipogenic

transcriptional program [26].

The results of a study carried out on obese prone

(OP) rats [27], revealed that high-salt intake induced

an increase in the size of WAT but a reduction in

number of adipocytes, accompanied by twofold

increase in circulating leptin. It was suggested that

high sodium chloride content diet could modulate

leptin levels, independently of obesity. In accordance

with this recently conceived concept regarding leptin

physiology [28], rodents treated with a high salt (HS)

diet were resistant to metabolic effects of leptin: as Wb,

and adipose mass-reducing [29, 30], but leptin’s

sympatho-excitatory actions still remains intact. The

accumulated knowledge so far brings us to test the

follow hypothesis: if leptin, vasopressin and

aldosterone are involved in breeding regulation of

desert-adapted rodents than the three hormones will

have a direct effect through their receptors on the

gonads or indirect effect of these hormones via WAT.

In order to establish the metabolic status of the tested

mice we also detected serum levels of free fatty acid

(FFA) and leptin in males and females of a desert

adapted population of common spiny mice, A.

cahirinus acclimated to short day (SD) or long day

(LD) photoperiods.


Studied mice were recruited from a desert adapted

laboratory colony of A. cahirinus, maintained at

Oranim campus, University of Haifa. Mice in the

colony are descendants of individuals originally

captured on the western Dead Sea shores. All tested

mice, at the beginning of acclimation, were adults,

aged four months. In the colony, mice were

maintained at an ambient temperature of 26 ± 2 °C

under a photoperiod regime of 12L:12D. In order to

avoid bias results related to Wb, both SD and LD

acclimated groups had a similar average Wb (34-36 ± 3

g) at the beginning of experiments. Ethical clearance

for the use of animals was provided by The Ethical

Committee, University of Haifa [8].

2.1 Acclimation to Increasing Salinity under Different

Photoperiod Regimes

36 individuals (18 females and 18 males) were

acclimated to short days (SD, 8L:16D, lights on from

07:00 a.m.) while 26 individuals (12 females and 14

males) were acclimated to long days (LD, 16L:8D,

lights on from 07:00 a.m.) inside a controlled

environmental cabinet (158 × 77 × 74 cm; Meditest

600/1300, Austria) for three weeks. As these

individuals were used also in for measuring other

variables for further information on acclimation

groups [8].

2.2 Sacrificing Animals and Collecting Samples

At the end of acclimation to the highest dietary

salinity (5% NaCl) mice were, anesthetized by

injecting a cocktail of Ketamin (10 mg/kg) and

Rampoone (100 mg/kg), and then sacrificed by

decapitation. All animals were sacrificed between

10:00 and 12:00 as reported earlier for our studied

individuals [8]. (For both photoperiod groups three

hours after lights were on). Blood samples were

collected and centrifuged at 3,000 rpm for 10 min.

Serum was collected and stored at -20 °C for

analyzing serum leptin hormone level and FFA.

WAT and gonads were removed and weighed. The

different organs mass was determined by using an

analytical scale (1907 MP8 Sartorius, Germany) and

calculated as percent of Wb. Organs were stored at

-20 °C in RNA later (Beit Haemek, Israel) for RNA

stability.

2.3 Determination of Serum Leptin Levels

Serum leptin levels were measured by using a

commercial ELISA (R&D System, USA) kit


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according to manufacturer’s instructions. This assay

employs the quantitative sandwich enzyme

immunoassay technique. The product of these

enzymatic reactions determined

spectrophotometrically, by ELISA reader (Power

Wave XS, BioTek Gen 5) at a wavelength of 450 nm.

2.4 Determination of Serum FFA Levels

In the assay used, fatty acids are converted to their

CoA derivates, which are subsequently oxidized with

the concomitant generation of color/fluorescence. C-8

(octanoate) and longer fatty acids can be easily

quantified by colorimetric (570 nm) method with

palmitic acid employed as a standard (Free Fatty Acid

Quantification Kit, BioVision, USA) according to

manufacturer’s instructions.

2.5 Total RNA Extraction and Reverse Transcription

The reverse transcription-polymerase chain

reaction (RT-PCR) has become a standard tool in

quantification of gene expression analysis studies [31].

Total RNA was extracted from mice testis using

RNeasy Mini Kit (Qiagen, Germany). For mRNA

purification from testis and RNA extraction from

WAT the authors used EZ-RNA kit (Biological

Industries, Israel, Beit Haemek Ltd.). Isolated total

RNA was quantified photometrically at a

wavelength of 260 nm. Quality of RNA was verified

by loading 1 μL of total RNA onto a RNA 6,000

Nano Chip using the Nano Assay 600 Bioanalyzer

(Agilent, Waldbronn, Germany) following the

manufacturer’s instructions. Single stranded cDNA

was generated out of 0.5 μg total RNA using a High

Capacity cDNA RT Kit (Applied Biosystems, UK).

The obtained cDNA was stored (-20 °C) for further

analysis.

2.6 Receptors Detection

Testis and WAT were tested for the expression of

aldosteron (Nr3c2: For reference we used NCBI

sequence NM_001083906.1) and vasopressin (AVPr:

For reference we used NCBI sequence NM_016847.2)

receptor genes. The expression of leptin receptor was

measured only in gonads (Ob-Rt: For reference we

used NCBI sequence NM_146146.2). For reference

housekeeping gene GAPDH (glyceraldehyde-3-

phosphate dehydrogenase: NM_008084.2 as reference

sequence) was used.

2.7 Primer Design

All primer sets on exon-exon junction site, were

designed to have a Tm of approximately 60 °C, to

have a GC content of approximately 50%, and to

generate a PCR amplicone less than 150 bps. Finally,

BLAST searchers were performed on primer pair

sequences using the NCBI database to check for

uniqueness. Primer sets and identifiers are provided

(Appendix 1).

2.8 qRT-PCR Amplification

qRT-PCR (quantitative real-time reverse

transcription-PCR) has become the benchmark for the

detection and quantification of RNA targets.

Quantitative real-time PCR (qRT-PCR) was

performed using SYBR Green technology [31];

primers were designed by Sigma, Israel. For the

real-time PCR procedure, gene-specific primers and

SYBR Green Real-Time PCR Master Mix (T Applied

Biosystems, CA, USA) were used. Relative gene

expression was detected by the ABI Prism 7000

Genetic Analyzer (Applied Biosystems, CA, USA).

The relative expression (relative quantification: RQ)

of each target gene (Ob-Rt; AVP; Nr3c2 genes), was

normalized to the amount of GAPDH as housekeeping

gene transcript in the same cDNA. RQ relates the

PCR signal of the target transcript in a treatment

group to that of another sample such as an untreated

control. A melting curve analysis was performed to

verify that a single PCR product was generated.

Negative controls, performed by omitting reverse

transcriptase from the RT step, remained consistently

negative.


1086

2.9 Statistical Analysis

All values are given as mean ± SEM, measured

values of control and experimental groups were

compared using two-tailed independent t test on SPSS

15.0.1 for Windows. For group comparison one way

ANOVA was used. Post-Hoc test was conducted

using LSD. Difference in mean values were

considered significant when P < 0.05. Actual

probability values were given for each comparison.


The metabolic status of SD- and LD-acclimated

males and females (control) as well as ST-males and

females was estimated from serum FFA and leptin

concentrations. The relation between WAT as

metabolic and endocrine tissue is assessed from the

correlation between FFA and leptin (Figs. 1 and 2).

In order to assess these relations with the

reproductive system, the response to increased salinity

in diet is evaluated from the mRNA receptors

expression of Nr3c2, AVP and Ob-Rt in the gonads

(Figs. 3 and 4).

In addition, the role of photoperiod effect on

metabolic and reproduction molecular response was

highlighted, in both sexes of desert-adapted A.

cahirinus population by studying receptors activation

in the gonads.

Because of massive reduction in Wb, and WAT

mass as result of salinity treatment, WAT could only

be collected from LD and SD control groups mRNA

receptor expression using our primers only

Aldosterone, but not AVP was detected. However, no

significant differences between the two photoperiod

acclimated groups were noted.

3.1 Female Responses to Photoperiod Manipulations

and ST Treatment

FFA levels were significantly elevated in

ST-LD-acclimated group, compared with the

ST-SD-mice (P < 0.05; F1,11, = 4.589). ST caused a

decrease in serum leptin levels in both, LD- and

SD-acclimated females, compared with their control

groups (P < 0.05). There was a trend to increase leptin

levels (~ 25%) in SD-acclimated females, compared

with the LD-females (Table 1A). However, FFA

levels in LD- and SD-acclimated (control) females

were similar. There was a strong positive correlation

(R2 = 0.48, P < 0.05) between leptin and FFA serum

levels in SD-acclimated females (Fig. 1).

As ovaries of ST-LD-acclimated females were

atrophied we could only compare between ovaries of

SD- and ST-SD-females. Expression of Nr3c2

mRNA was not affected by ST in SD-acclimated

females while, a strong effect of ST was noted in

Fig. 1A Correlations between serum FFA levels (nmol/µl) and leptin levels (pg/mL) of control long day acclimated (LD control) and salinity treated (LD-ST) desert-adapted female common spiny mice Acomys cahirinus. (n = 6 in each group). R2 = 0.26 for LD control group; R2 = 0.03 for LD-ST group.


1087

Fig. 1B Correlations between serum FFA levels (nmol/µL) and leptin levels (pg/mL) of control short day acclimated (SD control) and salinity treated (SD-ST) desert-adapted female common spiny mice Acomys cahirinus. R2 = 0.48, *P < 0.05 (Pearson) for SD-control group, R2 = 0.27 for SD-ST group.

Fig. 2A Correlations between serum FFA levels (nmol/µL) and leptin levels (pg/mL) of control long day acclimated (LD control) and salinity treated (LD-ST) desert-adapted male common spiny mice Acomys cahirinus. R2 = 0.63; *P < 0.01 (Pearson) for LD-control group, R2 = 0.51, *P < 0.05 (Pearson) for LD-ST group.

Fig. 2B Correlations between serum FFA levels (nmol/µL) and leptin levels (pg/mL) of control short day acclimated (SD control) and salinity treated (SD-ST) desert-adapted male common spiny mice Acomys cahirinus. R2 = 0.16 under SD-acclimation. R2 = 0.65, *P < 0.01 (Pearson) for SD-ST group.


1088

Fig. 3 Receptors mRNA expression (relative quantification—RQ) in ovaries of short day (SD control) acclimated and salinity treated (SD-ST) desert adapted female common spiny mice A. cahirinus. Nr3c2: aldosterone receptor gene expression; AVP: vasopressine receptor gene expression; Ob-Rt: leptin receptor gene expression. *P < 0.05 in SD-ST females, compared with SD-control ones.

Fig. 4 Receptors mRNA expression (Relative quantification—RQ) in testis of short and long day (SD, LD-control) acclimated and salinity treated (SD-ST, LD-ST) desert adapted female common spiny mice A. cahirinus. Nr3c2: aldosterone receptor gene expression; AVP: vasopressine receptor gene expression; Ob-Rt: leptin receptor gene expression. *P < 0.05 in treated groups, compared with their controls.

Table 1A Leptin and FFA concentration in serum (± SEM) of short and long day acclimated (SD, LD-control) and salinity treated (SD-ST, LD-ST) desert-adapted female common spiny mice Acomys cahirinus.

Treatment Leptin levels (pg/mL) FFA levels (nmol/µL)

LD female 2,700 ± 120 0.045 ± 0.01

LD-ST female 2,000 ± 80* 0.078 ± 0.015*

SD female 3,600 ± 100 0.045 ± 0.02

SD-ST female 2,600 ± 95* 0.042 ± 0.015*

Leptin (pg/mL) and FFA (nmol/μL) concentration (n = 9 in each SD group) and (n = 6 in each LD group). Leptin levels in LD-ST and SD-ST females compared with their control *P < 0.05. FFA serum levels in LD-ST females compared with their control, *P < 0.05. FFA serum levels in SD-ST females compared with the LD-ST ones *P < 0.05, (F1,12 = 1.34).

Table 1B Leptin and FFA serum levels (± SEM) of short and long day acclimated (SD, LD control), SD and LD salinity treated (SD-ST, LD-ST) desert adapted males of common spiny mice Acomys cahirinus.

Treatment Leptin levels (pg/mL) FFA levels (nmol/µL)

LD male 3,500 ± 115 0.04 ± 0.01

LD-ST male 2,300 ± 100* 0.072 ± 0.01*

SD male 3,200 ± 178 0.04 ± 0.01

SD-ST male 2,400 ± 89* 0.091 ± 0.015*

Leptin (pg/mL) and FFA (nmol/μL) concentration (n = 9 in each SD group) and LD (n = 6 in each LD group). Leptin levels in LD-ST and SD-ST males compared with their control, *P < 0.05. FFA serum levels in LD-ST and SD-ST males compared with their control *P < 0.05.

RQ

R

Q


1089

mRNA receptors expression genes of AVP and Ob-Rt,

which were significantly (P < 0.05) increased in

ST-SD females compared with their controls (Fig. 3).

3.2 Male Responses to Photoperiod Manipulations

and ST Treatment

No effect of photoperiod was noted, as under both

LD and SD acclimation FFA serum levels were

similar. However, ST caused a significant (P < 0.05)

increase in serum FFA and a significant decrease (P <

0.05) in leptin levels, in both, LD and SD group

compared with their control groups (Table 1B). The

correlations between leptin and FFA serum levels

were significant and negative for both, ST-LD (R2 =

0.51, P < 0.05) and ST-SD (R2 = 0.65, P < 0.01) mice.

The same correlation was noted as positive and

significant (R2 = 0.63, P < 0.01) in LD-acclimated

mice (Fig. 2).

The mRNA receptor expression of Nr3c2 was affected

by ST in both, LD- and SD- males. A significant (P <

0.05) increase in mRNA receptor expression genes

was noted in both Nr3c2 and AVP under both

photoperiod regimes. However, Ob-Rt mRNA

receptor expression genes increased significantly (P <

0.05) by ST only under LD-acclimation. ST under

SD-acclimation had no effect on testis Ob-Rt mRNA

receptor expression genes (Fig. 4).

4. Discussion

4.1 Metabolic Response to ST

According to the results of previous studies, ST

induces a dramatically Wb decrease [4, 5] in

desert-adapted A.russatus. A massive reduction in Wb

and WAT mass was noted in ST-SD males where Wb

decreased significantly (P < 0.001) by 26% ± 0.1%,

while in ST-LD males the loss was only of 13% ±

0.03% (P < 0.01). In SD-ST females a Wb loss was of

only 17% ± 0.03% but still significant (P < 0.01),

while for LD-ST females a 3% ± 0.2% significant (P

< 0.01) decrease in Wb from their initial values as

reported earlier [8] for A. cahirinus, individuals used

in the present study. Some other previous studies

showed that dietary sodium chronic restriction has

been related to increased WAT mass in rats [32].

WAT development depends on a balance between

food consumption and energy expenditure [33]. It was

also revealed that high salt dietary consumption

significantly decreased feed intake in sheep [9, 34].

The results of our study, in addition to the progressive

decrease in Wb, revealed an almost complete

abolishment of WAT in ST-mice as reported earlier [8].

WAT reduction and Wb values decrease in our study

could not be explained by reduced energy intake, as

ST mice consumed the same amount of food as the

control mice throughout the experimental period [8].

We tested adipocytes lipid mobilization resulted from

ST compared with their control. This process known

as lipolysis, consists of Triacylglycerole (TAG)

hydrolysis, FFA and glycerol release, which represent

an important mechanism for controlling WAT mass

and metabolism [35]. ST males showed an increase in

FFA release, during both, LD and SD acclimation

compared with their controls. ST-LD-acclimated

females showed increased FFA release compared with

ST-SD females (Table 1A). Chronic salt loading may

increase dysfunction of fat cells in lean and obese

alike [35]. A significant linear positive relationship

between leptin levels and WAT was noted in previous

study where lower serum leptin levels in cold

acclimated animals could act as a starvation signal [36].

It was also shown that ST induced hyperleptinemia

and it may stimulate the lipolytic process by a direct

action [37].

High salt (3% NaCl) treated rats exhibited higher

plasma leptin levels compared with those of controls [38].

Higher plasma leptin levels were also reported for rats

kept on a 4% NaCl diet for 10 weeks compared with

those kept on 0.8% NaCl [27]. In our study, ST caused

hypoleptinemia in both, males and females, under

both, LD and SD-conditions compared with their

controls. In addition, the correlations between leptin


1090

and FFA levels were significant, in both, males and

females (Figs. 1 and 2). These correlations were

positive in LD and SD-acclimated groups (controls),

but negative, in ST groups. Combining our results

with Ref. [35], we suggest that ST may alter (directly

or indirectly) the secretion function and metabolic

activity of WAT.

4.2 Reproductive Response to ST

Previous studies on leptin receptors (leptin-R) in

rodents have demonstrated the expression of leptin-R

gene in the hypothalamus, ovary, uterus, testis and

pituitary by reverse transcriptase polymerase chain

reaction (RT-PCR) [39]. In our study using the same

method we detected a significant increase in

expression levels of mRNA receptors genes for

aldosterone (Nr3c2) in testis of ST-males under the

two photoperiods (P < 0.05, Fig 4). ST caused a

significant increase (P < 0.05, Fig. 4) in expression

level of vasopressin (AVP) mRNA receptor genes in

testis under both photoperiods while for females only

SD-ST could be measured (P < 0.05, Fig 3). High

gene expression levels of leptin (Ob-Rt) mRNA

receptors were noted in SD-ST females (P < 0.05,

Fig 3) while in males only in LD-ST individuals (P <

0.05, Fig. 4), compared with their controls. We

suggest that ST caused a significant decrease of leptin

levels in serum and for this reason; leptin receptors

sites expressed on gonads were not occupied by the

hormone.

The authors have no data on receptors gene

expression in ovaries of ST-LD females, as there

ovaries were atrophied. Yet the authors can suggest

that ST had an effect on metabolism and reproductive

ability of ST-LD-acclimated females, as leptin levels

were decreased under both photoperiod acclimations

regimes. It is important to note that ingestion of

salt has a dramatic effect on sheep reproductive

capacity [6].

Aldosterone and vasopressin are two essential

hormones for controlling the water balance and

osmoregulation [40] on the one hand and they affect

the reproductive ability of desert adapted rodents on

the other [8]. The AVP gene is now known to be

expressed in a number of peripheral organs such as the

adrenal glands, ovaries, and testes [41]. Ivell [42]

reported that AVP mRNA is detectable in the rat testis.

It is now well documented that aldosterone receptor

are also expressed in non-epithelial tissues, including

the cardiovascular and central nervous systems as well

as on the white adipose tissue [43].

The results of our study show that a part of the

involvement of these two hormones in osmoregulation,

they are also possibly involved in inhibiting

reproduction under osmolarity stress as there serum

levels increased also mRNA receptor expression genes

increase. These increases suggest an involvement of

molecular mechanism in the gonads as a response to

increased osmolarity stress.

Human WAT cells secrete mineralocorticoid-

releasing factors [44]. The AVP receptors (V1b and

V2) were attributed in mice to lipid metabolism;

expression of the two genes was also noted in the

heart, liver, kidney, skeletal muscle, BAT, and WAT.

The V1a receptor was expressed in all tissues

examined, but the V1b receptor was expressed only in

WAT, while the V2 receptor in mice was expressed

only in the kidney [45]. We searched for AVP and

Nr3c2 mRNA receptor genes expression in WAT. For

expression of receptors we had compared only SD-

and LD-acclimated control males, as WAT was

abolished due to ST. There was no detectable AVP

mRNA receptor gene expression, but only of Nr3c2.

There was no significant difference in expression

levels between LD- and SD-acclimated groups.

5. Conclusions

The data emerging from this study support our

general concept that: successful breeding in

desert-adapted rodents depends on two different

environmental signals namely: (1) photoperiod as an

initial cue, (2) availability of sufficient water and food


1091

resources in the environment, as an ultimate cue. The

idea of two signals was first suggested by Louw and

Seely [46]. Therefore, our results on the endocrine and

molecular levels are of importance for the proposed

idea as they show that hormones regulating water

balance and osmoregulation (vasopressin, aldosterone)

as well as energy storage (leptin) through receptors

presented on the gonads are involved in reproduction

of desert-adapted rodents. Together, with our earlier

results [8] we can conclude that photoperiod on the

one hand and availability of water and food resources

on the other are involved in the comprehensive

activation of the reproductive system in the desert

adapted population of A. cahirinus.

Acknowledgments

The authors thank the ISF (Israel Academy of

Science and Humanities) for financial support through

a grant to Abraham Haim and Fuad Fares. We also

would like to thank Ms. Lilach Ashkenazi for her help

and constructive comments. We thank Ms. Nina

Dinov and the staff of the department of Biology at

Oranim Campus for their assistance in maintaining the

animals. Authors also thank anonymous advisors for

their constructive comments on an earlier version of

this paper.

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Appendix 1

GenBank: Genetic sequence database at the National Center for Biotechnical Information (NCBI): GenBank ID Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) NM_008084.2

GAPDH-F: 5’-AGGTCGGTGTGAACGGATTTG-3’; GAPDH-R: 5’-TGTAGACCATGTAGTTGAGGTCA-3’;

Aldosterone receptor gene (Nr3c2) NM_001083906.1 Nr3c2-F: 5’-GAAGAGCCCCTCTGTTTGCAG-3’; Nr3c2-R: 5’-TCCTTGAGTGATGGGACTGTG-3’;

Vasopressin receptor gene (AVP) NM_016847.2 AVP-F: 5’-CAATTTCGTTTGGACCGATTC-3’; AVP-R: 5’-GGGTTGCAGCAGCTGTTCA-3’;

Leptin receptor gene (Ob-Rt) NM_146146.2 OB-Rt-F: 5’-GTC TTC GGG GAT GTG AAT GTC-3’; OB-Rt-R: 5’-ACC TAA GGG TGG ATC GGG TTT-3’.


Influence of Abiotic Elicitors on Accumulation of Thymol

in Callus Cultures of Origanum vulgare L.

Abedaljasim M. Jasim Al-Jibouri, Ashwaq S. Abd, Duha M. Majeed and Eman N. Ismail

Biotechnology Research Center, Al-Nahrain University, Baghdad 10072, Iraq

Received: May 05, 2012 / Accepted: July 20, 2012 / Published: October 30, 2012.

Abstract: Callus cultures of Origanum vulgare L. were established from leaf discus on Murashige and Skoog (MS) medium containing different levels of growth regulators, i.e., 2,4-Dichlorophenoxyacetic acid (2,4-D), Naphthalene acetic acid (NAA), Benzyl Adenine (BA) and Kinetin (Kn) and incubated under dark condition. Callus tissues were employed to study the influence of abiotic elicitors on the production of thymol. Constant weights of callus (300 mg) were cultured on accumulation medium treated separately with each one of elicitors used (50 g/L sucrose, 200 mg/L NaCl and 50 or 100 mg/L proline). The fresh and dry weights of callus were recorded after six weeks. The result indicated that maximum production of fresh and dry callus weight were 1,014 mg and 46.20 mg respectively achieved at 0.5 mg/L 2,4-D and 3 mg/L BA adding to the medium. Dry callus tissues were extracted with 70% methanol and analyzed by HPLC to determine the concentrations of thymol. The addition of abiotic elicitors to MS medium caused significant reduction in fresh weight of callus compared with control treatment. The concentration of thymol in the callus cultured on control treatment was 146.6 ppm. The data showed that 50 or 100 mg/L proline produced the highest yield of thymol 181.48 ppm and 174.58 ppm respectively, followed by sucrose 162.9 ppm, whereas the treatment with NaCl caused reduction in thymol concentration to percentage of 50.56% compared with the control. Key words: Origanum vulgare L., thymol production, callus culture, abiotic elicitors.

1. Introduction

Origanum vulgare L. is a member of the

Lamiaceae family (Labiatea), commonly named

oregano, wild marjoram, marzanjosh or mardaqoush,

which grows abundantly on stony slopes and in

rocky mountain areas at a wide range of altitude [1].

The Origanum species, which are rich in essential

oils, have been used for thousands of years as spices

and as local medicines. Arial parts of Origanum

vulgare are used in respiratory tract disorders such as

cough or bronchial catarrh (as expectorant and

spasmolitic agents), in gastrointestinal disorders (as

choleretic, digestive, eupeptic and spasmolitic agents)

as oral antiseptic, in urinary tract disorders (as

diuretic and antiseptic) [2] and modulates blood

Corresponding author: Abedaljasim M. Jasim Al-Jibouri,

Ph.D., assistant professor, research field: plant biotechnology. E-mail: [email protected].

sugar and lipids [3]. The main components of

essential oil of Oregano plants are thymol, carvacrol,

p-cymen and β-pinen [1, 4, 5]. Essential oil is a

mixture of volatile compounds produced in small

quantities as secondary metabolites from aromatic

and medicinal plants [6]. Thymol is a most valuable

crystalline phenol [7], have many biological

activities, i.e. anti-oxidant [8], anti-cancer [9, 10],

anti-mutagenic [11], anti-inflammatory [12],

anti-microbial [13, 14], and anti-fungal activity [15].

The accumulation of secondary metabolites in plant

tissue cultures has been obtained from various

medicinal plants. The production of hyoscyamine and

scopolamine has been reported in Callus cultures of

Datura metel L. [16]. High in vitro production of

indole alkaloids from Catharanthus roseus tissue

culture has also been reported [17]. In vitro studies in

Lamiaceae, callus culture of Origanum species were

Influence of Abiotic Elicitors on Accumulation of Thymol in Callus Cultures of Origanum vulgare L.

1095

induced to study the effect of different type of nutrient

media and various growth regulators on different

morphological responses [1], Arafeh et al. [6] studied

the content of essential oil in intact plants (in vitro and

ex vitro), callus, and cell cultures in Origanum vulgare

and O. syriacum. The production of volatile oils

has been shown in callus culture of Origanum

vulgare [18].

The advantages of producing secondary compounds

using tissue culture technique are more reliable,

simpler, predictable, easy isolation and efficient

compared with in vivo production. In addition, cell

culture can yield a source of defined standard

phytochemicals in large volume and as technique

could be a good model to evaluate various elicitors

effects.

Secondary metabolite synthesis may be enhanced in

vitro by controlling the composition of the culture

medium and the environment, certain secondary

compounds are produced by the plant under the

influence of various biotic and abiotic factors that are

known as “elicitors” [19].

The main objective of present study was to examine

the effect of exogenous elicitors on biomass

production and accumulation of thymol in callus

tissue of Origanum vulgare L. under dark condition.


2.1 Plant Material

The plants of Origanum vulgare L., were collected

at the flowering stage in April 2010 from the

Botanical Garden of Biotechnology Research Center,

Al-Nahrain University, Baghdad, Iraq.

Taxonomic identification of plant material was

conferred by Prof. Dr. Ali Al-Musawi, Department of

Biology, Baghdad University. Leaf explants were

taken and washed with tap water for 30 min, then

immersed in 3% of sodium hypochlorite and added

2-3 drops of Tween 20 for 10 min, finely these leaves

were washed once with 70% ethanol and several times

with distilled sterile water.

2.2 Callus Induction and Maintenance

Sterilized leaf segments (0.5 cm2) were cultured on

solidified MS medium [20] supplemented separately

with 0.5 mg/L or 1 mg/L 2,4-D, 0.5 mg/L or 1 mg/L

NAA, 0.5 mg/L 2,4-D and 3 mg/L BA and 0.5 mg/L

2,4-D and 3 mg/L Kn. All cultures were performed in

the dark condition at 25 ± 2 °C. Data were recorded

after 6 weeks on fresh and dry weight of callus. The

callus production was selected and maintained on

fresh media (supplemented with 0.5 mg/L 2,4-D and 3

mg/L BA which produced highest yield of callus) to

study the effect of abiotic elicitors on thymol

production.

2.3 Abiotic Elicitors Used

Equal amount of callus (300 mg) was cultured on

maintenance medium (0.5 mg/L 2,4-D and 3 mg/L

BA), three abiotic elicitors have been added to

medium separately, 50 mg/L sucrose, 200 mg/L

sodium chloride (NaCl) and amino acid proline at two

concentrations 50 mg/L or 100 mg/L. All cultures

were incubated under dark condition at 25 ± 2 °C.

Fresh and dry weights of callus were recorded after 6

weeks of incubation and thymol content were

determined.

2.4 Thymol Extraction

Dry callus (200 mg) of each treatment were

grounded and macerated over night in 10 mL of 70%

methanol at room temperature with periodical mixing,

then filtrated and concentrated at 45 °C to get 5 mL of

sample. All samples were filtrated through a 0.22 µm

Millipore filter and then stored in dark at 4 °C until

used [21, 22].

2.5 HPLC Analysis

Thymol contents were determined using HPLC

instrument (Cecil Company, England). ODC (C18)

column (25 cm × 4.6 mm, partial size 5 µm) was used

with mobile phase acetonitrile-water (40:60; v/v),

flow rate of 1.0 mL/min. The detection was carried


1096

out at 254 nm. The quantitative determination was

carried out through the use of external standard

method.


All experiments were done with minimum of 20

replicates per treatment. Significance of treatment

effect was determined by using analysis of variance

(ANOVA) followed by LSD test (P ≤ 0.05) to

determine significant differences among treatment

means.

3. Results and Discussion

3.1 Callus Induction

The results showed that significant differences

between the growths regulators in fresh and dry

weight of callus produced from leaf explants of O.

vulgare (Fig. 1). The supplementation of MS medium

with 0.5 mg/L 2,4-D and 3 mg/L BA gave the

maximum average of fresh and dry weights of callus

compared with other supplementations, reached 1014

mg and 46.20 mg respectively which significant

differences of all treatments except the treatment of

0.5 mg/L .This callus was healthy, white in color and

compact in texture. Medium supplemented with 0.5

mg/L 2,4-D also showed high callus weight but it was

gelatinous in texture. The control treatment gave

lowest fresh and dry weight of callus (118 mg and

16.5 mg) respectively which significant differences

with all treatment. The obtained results were in

agreement with El-Gengaihi et al. [1] who reported

that supplementation of auxin and cytokinin (0.5 mg/L

NAA and 3 mg/L BA) to MS medium gave the best

results of callus induction form Origanum species.

Similarly to the authors’ results, Stojakowska et al. [23]

reported that callus tissue of Inula helenium was

obtained from leaf explants when cultured on

solidified MS medium containing 1 mg/L 2,4-D and 3

mg/L Kn. Arafeh et al. [6] mentioned that best callus

induction in O. vulgare was obtained at lower levels

of 2,4-D (0.5 mg/L or 1 mg/L) added to medium. The

result appeared that callus initiation and production

were dependent on the presence of auxin and

cytokinin, which stimulate both cell division and cell

elongation [24].

3.2 Effects of Abiotic Elicitors

The result in Fig. 2 illustrated the adding of abiotic

elicitors such as 50 mg/L proline to medium produced

highest average of fresh and dry weight of callus

reached 1,357 mg and 110.4 mg respectively, which

are significantly differences with all treatments except

0

200

400

600

800

1000

1200

118

978885

750 728

1014

862

16.5 43.14 42.5 41.7 39.7 46.2 39.8

wei

ghts

mg

Plant regulatores

Fresh weight

Fig. 1 Effect of growth regulators on fresh and dry weight of Origanum vulgare L. grown on MS solid medium for 6 weeks. LSD at (0.05) level for fresh weight: 83.8. LSD at (0.05) level for dry weight: 5.4.


1097

Fig. 2 Influence of abiotic elicitors on fresh and dry weight of callus culture for Origanum vulgare L. grown on MS solid medium for 6 weeks. LSD at (0.05) level for fresh weight: 180.1. LSD at (0.05) level for dry weight: 14.54.

Table 1 Influence of abiotic elicitors on thymol content in callus culture of Origanum vulgare L.

Type of elicitors Thymol con. (ppm) Percentage of control

positive Negative

Control 146.6 - -

Sucrose 50 g/L 162.99 11.18

NaCl 200 mg/L 72.50 - 50.56

Proline 50 mg/L 181.48 23.79 -

Proline 100 mg/L 174.58 19.09 -

the control which produced 1,303 mg and 97.4 mg of

fresh and dry weight of callus respectively. The

addition of NaCl (200 mg/L) to the medium produced

lowest fresh and dry weight of callus (1,044 mg and

88.2 mg, respectively). The reduction of callus

production after adding abiotic elicitors, except 50

mg/L proline, might be attributed to the stress in the

medium by these agents on cell growth and cells

division. The results obtained are in agreement with

Razmjoo et al. [24] who found that increasing of

salinity and drought stress decreased almost all growth

parameters of Matricaria chamomila and also with

result of Ajungla et al. [19] were used biotic and

abiotic elicitors in cell culture of Datura metel.

About the effect of abiotic elicitors on thymol

concentration in the callus, the result showed in

Table 1, difference concentration of thymol essential

oil produced from callus extract of O. vulgare depending

on type and concentration of abiootic elicitors used

(Fig. 3). Concentration of thymol was increasing in

the callus grown on medium containing 50 mg/L or

100 mg/L proline or 50 gm/L sucrose which reached

to level 181.48 ppm, 174.58 ppm and 162.99 ppm

with percentage increase of 23.79%, 19.09% and

11.18%, compared with the control (146.60 ppm). The

results also indicated that supplementation with other

abiotic elicitors such as sodium chloride (200 mg/L)

caused reduction in thymol concentration to

percentage of 50.56% compared with the control.

Previous investigations found that adding biotic and

abiotic elicitors to medium cultures effect either

positive or negative on growth of callus and secondary

metabolic compounds production depending on type

and concentration of elicitors [19]. Adding abiotic

elicitors to the medium such as jasmonic acid and

SiO2 with different concentrations caused an increase

the amounts of secondary metabolic compound of

Ammi majus in callus cultures [25]. The highest


1098

Fig. 3 HPLC chromatograms for: A: standard thymol; B: control (without elicitors) C, elicitor treatment (proline 50 mg/L).

essential oil produced from chamomile plant treated

by proline at concentration 50-150 ppm compared

with the control [26]. The results indicated in this

investigation that proline is favorable for promoting

the accumulation of thymol essential oil in callus of O.

vulgare incubated in dark condition.

4. Conclusions

As a conclusion, the results indicated the possibility

of producing callus from Origanum vulgare L., and

that addition of abiotic elicitors to the medium

increase secondary metabolites (thymol)

concentrations in callus. Thus there is a clear in using

tissue culture techniques for large scale and

continuous production of targeted secondary

metabolites.

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Low Peptone Dose as Inductor of Alkaline Protease

Promoter Used for Invertase Gene Expression in

Yarrowia lipolytica

Łukasz Śnieżewski1, Ewa Walczak2, Zbigniew Lazar3 and Małgorzata Robak3

1. Department of Tumor Immunology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław

53-114, Poland

2. Department of Medicine, The Witelon University of Applied Sciences, Legnica 59-220, Poland

3. Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, Wroclaw 50-375,

Poland

Received: August 01, 2012 / Accepted: August 06, 2012 / Published: October 30, 2012.

Abstract: According literature, the induction of Yarrowia lipolytica alkaline protease promoter (PXPR2) is efficient in pH > 6.0 and with high peptone dose. To establish optimal pH and peptone concentration for induction of invertase gene (suc2 of Saccharomyces cerevisaie) under PXPR2 in new Y. lipolytica A-101 invertase positive (Suc+) transformants their growth on Bioscreen C was analyzed. Minimal mineral medium with thiamine (MMT) and sucrose (1%), adjusted to pH from 5.8 to 7.6 and supplemented by 0-0.1% of peptone was used. Biomass (OD), maximal specific growth rate (µmax) and consumed sucrose were measured. Maximal yeasts growth, resulting from the optimal PXPR2 induction, was observed at pH 7.2 and with very low peptone doses (0.0025% and 0.01%). For five clones (A-101 B56-5; A-101 B54-6; A-101 B57-4; A-101 A18 and W29 ura3-302) only 0.005% of peptone was needed. Amount of hydrolyzed sucrose varied from 24% to 83% and µmax from 0.06 to 0.28 h-1. Suc+ clones differ in growth parameters, so the site of yeast cassette integration into genome influences expression level of suc2 under PXPR2. Designing large scale processes with Y. lipolytica Suc+ clones peptone concentration has to be 100 times smaller than recommended so far. Key words: PXPR2 induction, Bioscreen C, Suc+ transformants, Yarrowia lipolytica, invertase.

1. Introduction

Extracellular alkaline protease encoding gene (xpr2)

of Yarrowia lipolytica has 4,049 bp length

[http://www.ncbi.nlm.nih.gov] and encompasses

promoter which has over 700 bp [1]. The xpr2

promoter (PXPR2) was utilized in the construction of

many yeast expression cassettes allowing a variety of

heterologous genes to be expressed in Y. lipolytica [2-8].

Induction of PXPR2 is efficient in pH > 6.0

independently of metabolic signals [9-11] but some

authors claimed that peptone is needed for its optimal

Corresponding author: Małgorzata Robak, professor,

research fields: biotechnology and microbiology. E-mail: [email protected].

induction [7, 12]. Blanchini-Roland et al. [12] and

Madzak et al. [13] described some regulatory element

of PXPR2. The TATA box is on the position of -64 to

-59 from start codon and two UAS (upstream

activating sequences) were defined. UAS1 probably

was positioned between -800 and -769 nucleotide,

UAS2 between -145 and -127 bp. UAS1 encompasses

a sequence allowing binding of transcription factors.

UAS2 has a transcription factor binding site and a

homologous sequence to PacC, responsible for pH

induced expression of acide phosphatase in

Aspergillus nidulans [13]. One of the heterologous genes expressed in Y.

lipolytica under PXPR2 is suc2 from S. cerevisiae,

Low Peptone Dose as Inductor of Alkaline Protease Promoter Used for Invertase Gene Expression in Yarrowia lipolytica

1101

encoding invertase. Suc+ transformants of Y. lipolytica

were able to grow on sucrose (carbon source not

assimilated by wild strains) and were obtained by

Nicaud et al. [2], Mauersberger et al. [14], and Forster

et al. [7] as well as in the authors’ laboratory [15].

Obtaining homologous (Suc+ Ura–) and heterologous

(Suc+ Ura+) transformants allowed the author to study

PXPR2 induction by peptone and pH leading to suc2

expression in function of particular clones. The

authors have studied this gene expression by yeasts

growth characteristics in a very well defined mineral

medium with thiamine (MMT) and sucrose as sole C

source. The obtained results allowed the author to

determine optimal pH and minimal peptone

concentration needed for appropriated promoter

induction.


2.1 Yeasts

The 17 Suc+ transformants of Y. lipolytica (15

clones derived from Y. lipolytica A-101 and 2

references strains), as well as wild type (WT) used in

the study were listed in Table 1. All strains were

maintained on YPG at 4 °C and deposited at the

Culture Collection of Biotechnology and Food

Microbiology Department of Wrocław University of

Environmental and Life Sciences in Poland.

2.2 Media

Mineral medium with thiamine (MMT) containing

10 g/L of sucrose, supplemented with different doses

of peptone (0; 0.0025; 0.005; 0.01; 0.1; 1.0 g/L) and

of different pH (5.8; 6.2; 6.8; 7.2 or 7.6; 50 mM

phosphate buffer) was used. Detailed MMT

composition was described previously [16]. For Ura–

strains the medium was supplemented by the addition

of 20 mg/L of uracil.

Seed cultures for Bioscreen C (Oy Growth Curves

AB Ltd., Helsinki, Finland) studies were obtained

from flask growth cultures on MMT with 1% of

glucose (24 h, 28 °C, 180 rpm, New Brunswick

Scientific G10). Cultures were harvested (5,000 rpm,

Sigma 3-16K), washed twice with 0.9% NaCl,

suspended in 0.9% NaCl, incubated with shaking (180

rpm) for 2 h at 28 °C and standardized to OD = 0.23 –

0.25 (~ 1.8 × 106 cells/mL).

2.3 Culture Conditions and Analyses

Microcultures were performed in Bioscreen C

growth analyzer. In 200 microtubes, 300 µL of 1.17

concentrated MMT and 50 µL of defined seed culture

were placed. Each culture was prepared in 5

microtubes (= 5 repetitions). Cells growth in 28 °C for

72 hours was monitored by OD (420-580 nm),

measured every 15 minutes. Kinetics parameters of

growth determined as ODmax; lag phase duration and

specific growth rate (µmax) were calculated on the

basis of 5 parallel microtubes. Specific growth rate

was calculated according to Wilson et al. [17]

formula.

The concentration of glucose, fructose and sucrose

was measured in post culture media on Animex

Table 1 Yeast strains used in the studies.

Y. lipolytica strains Phenotype Provenance

W29 ura3-302 Suc+, Ura– INRA—Grignon, France, transformant of W29 (CLIB89; CBS7504)

H222-S4 Suc+, Ura– TUD—Drezden, Germany, transformant of H222

A-101 WT Suc–, Ura+ Soil isolate, Wrocław, Poland, wild strain used for transformation A-101 B54-6; A-101 B55-3; A-101 B57-4; A-101 A18 Suc+, Ura– Homologous transformants of A-101

A-101 B56-5; A-101 B58-2; A-101 B59-3; A-101 B60-4; A-101 B14-6; A-101 B7-6; A-101 B10A-5; A-101 B61-5; A-101 B62-5; A-101 KLON1; A-101 KLON10

Suc+, Ura+ Nonhomolous transformants of A-101


1102

HPX-87H column (0.6 mL/h; 0.01N H2SO4; room

temperature) by HPLC system with UV and RI

detector, as described previously [18].


The growth of selected Suc+ clones of Y. lipolytica

in MMT medium with sucrose and 0.1% of peptone

(as the first tested peptone dose) is presented on

Fig. 1. The growth of A-101 B56-5 and of reference

strains W29 ura3-302 was very good (OD > 1.5).

The growth of seven other clones was good (1 < OD

< 1.5), medium for next seven clones (0.7 < OD =

1.0) and residual for WT strain (OD < 0.4). The

growth rate of strains was different and maximal

specific growth rate ranged from 0.062 to 0.095 h-1

(Table 2).

In the next analysis, it was demonstrated that the

level of sucrose assimilation during the growth of 4

clones was only slightly affected by pH and was strain

specific (Fig. 2). Contrary to the growth, this

increased with rising pH and was rather independent

of the clones. Only at pH 5.8 a net differences in the

biomass accumulation by four strains were observed

(Fig. 3). Clone A-101 B54-6 consumed 62 % and

75 % of sucrose, respectively at pH 5.8 and 7.2. In the

same condition strain A-101 B59-3 consumed only

31% and 42% at respective pH values.

Subsequent microcultures analyses proved that the

dose of peptone for an efficacy PXPR2 induction was the

concentration of 0.01% independently of pH value

(Table 3). The µmax ranged from 0.0584 h-1 to 0.2807 h-1.

For suc+ Y. lipolytica A101 derived strains the maximal

Fig. 1 Bioscreen C growth of Suc+ Y. lipolytica strains on MMT medium with sucrose (1%) and peptone (0.1%); without (a) or with (b) uracil, respectively for heterologous and homologous recombinants.

(a)

OD

B56-5 B58-2 B59-3 B60-4 B14-6

B7-6 B10A-5 B61-5 B62-1

KLON1 KLON10 A-101 WT Medium control

B54-6 B55-3 B57-4

A18 W29 ura3-302

H222-S4

Medium control

OD

(b)

21.75

1.51.25

10.75

0.50.25

0

21.75

1.51.25

1

0.75

0.50.25

0


1103

Table 2 Bioscreen C growth of Y. lipolytica clones in MMT (pH 6.8) with sucrose ( 1%) and peptone (0.1%).

Y. lipolytica clone Biomass ODmax ± S.D. µmax (h-1)

A-101 B56-5 1.587 ± 0.0225 0.091 A-101 B58-2 1.262 ± 0.0334 0.069 A-101 B59-3 1.060 ± 0.0562 0.067 A-101 B60-4 1.177 ± 0.615 0.076 A-101 B14-6 1.341 ± 0.0646 0.078 A-101 B7-6 0.885 ± 0.046 0.089 A-101 B10A-5 0.844 ± 0.1241 0.077 A-101 B61-5 1.179 ± 0.157 0.077 A-101 B62-1 1.087 ± 0.0524 0.062 A-101 KLON1 0.949 ± 0.0932 0.077 A-101 KLON10 0.770 ± 0.1076 0.078 A-101 WT(a) 0.399 ± 0.0556 residual growth A-101 B54-6* 1.406 ± 0.0251 0.088 A-101 B55-3* 0.995 ± 0.0871 0.077 A-101 B57-4* 0.856 ± 0.0546 0.081 A-101 A18* 1.000 ± 0.0594 0.069 W29 ura3-302* 1.641 ± 0.0401 0.095 H222-S4* 0.872 ± 0.1223 0.077

*: with uracil (2%); (a): residual growth.

Fig. 2 Consumption of sucrose by Suc+ Y. lipolytica in function of medium pH. MMT with sucrose (10g/L) and peptone (0.01%): (1) A-101 B54-6; (2) A-101 B57-4; (3) A-101 B59-3; (4) W29 ura3-302.

Fig. 3 Bioscreen C growth of selected Suc+ Y. lipolytica strains on MMT with sucrose (1%) and peptone (0.01%), buffered to indicated pH values. (1) A-101 B59-3; (2) A-101 B54-6; (3) A-101 B57-4; (4) W29 ura3-302; (5) Medium control.

g/L

pH 5.8 pH 6.2 pH 6.8 pH 7.2 pH 7.6

(a)

(c)

(b)

(d)

(e)

pH 5.8 pH 6.2

pH 6.8 pH 7.2

pH 7.6

OD

21.751.5

1.251

0.750.5

0.250

21.751.5

1.251

0.750.5

0.250

21.751.5

1.251

0.750.5

0.250

21.751.5

1.251

0.750.5

0.250

21.751.5

1.251

0.750.5

0.250


1104

growth rate was noted at pH 7, 6 and for W29 ura3-302

at pH 6.8. At pH 7.2 all yeast strains accumulated more

biomass that in other pH medium values.

Choosing pH 7.2 as an optimal one for Bioscreen C

studies, the authors decided to check smaller peptone

concentration as an inducer of invertase gene

expression measured by the growth of nine clones.

Four of them (A-101 B59-3, A-101 KLON1, A-101

B55-3, H222-S4) did not grow when 0.005% of

peptone was in MMT (Fig. 4, only positive results

were shown). Five of all nine tested transformants

grew in the presence of 0.005% of peptone in the

medium, four of them even quite well. In the presence

of 0.0025 % of peptone, four clones have grown, two

with 0.001% and only one without peptone. However,

with decrease of peptone dose the diminution of

accumulated biomass and the elongation of lag phase

were observed, together with a smaller µmax (Table 4,

only positive results, of growth were presented, OD >

0.4).

Regarding sucrose consumption in a function of

small inductor doses (0.0025% and 0.005%), the best

growing clones (A-101 B56-5 and A-101 B54-6)

assimilated between 60%-83% of sugar added to the

medium (other clones have assimilated only about

20%). In the medium without peptone and with the

smallest dose (0.001%) sucrose assimilation by those

two clones drastically decreased to 18%-32% of the

initial amount (results not schown).

4. Discussion

Utilization of Bioscreen C, an automated

microbiological turbidimeter, for the study of

the yeast growth guarantees: the sterility during the

Fig. 4 Bioscreen C growth of selected Suc+ Y. lipolytica transformants on MMT with sucrose (1%), pH 7.2 and indicated peptone doses. (1) A-101 B56-5; (2) A-101 B59-3; (3) A-101 KLON1; (4) A-101 B54-6; (5) A-101 B55-3; (6) A-101 B57-4; (7) A-101 A18; (8) W29 ura3-302; (9) H222-S4; (10) Medium control.

(a) (b)

(c) (d)

2

1.75

1.5

1.25

1

0.75

0.50.25

0

2

1.75

1.5

1.25

1

0.75

0.50.25

0

2

1.75

1.5

1.25

1

0.75

0.50.25

0

2

1.75

1.5

1.25

1

0.75

0.50.25

0


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Table 3 Bioscreen C growth of selected Suc+ Y. lipolytica strains in MMT with sucrose (1%) and peptone (0.01%) at different pH.

Y. lipolytica strain pH Biomass ODmax ± S.D. µmax (h-1)

A-101 B54-6*

5.8

1.09 ± 0.074 0.0979

A-101 B57-4* 0.45 ± 0.017 0.1261

A-101 B59-3 0.49 ± 0.107 0.0584

W29 ura3-302* 0.91 ± 0.055 0.0828

A-101 B54-6*

6.2

1.25 ± 0.064 0.1276

A-101 B57-4* 1.05 ± 0.106 0.1224

A-101 B59-3 1.07 ± 0.048 0.0605

W29 ura3-302* 1.15 ± 0.105 0.1006

A-101 B54-6*

6.8

1.33 ± 0.077 0.0983

A-101 B57-4* 1.21 ± 0.074 0.1003

A-101 B59-3 1.23 ± 0.029 0.1075

W29 ura3-302* 1.38 ± 0.075 0.2807

A-101 B54-6*

7.2

1.41 ± 0.018 0.1925

A-101 B57-4* 1.49 ± 0.038 0.1738

A-101 B59-3 1.23 ± 0.057 0.1501

W29 ura3-302* 1.53 ± 0.131 0.2028

A-101 B54-6*

7.6

1.36 ± 0.166 0.24

A-101 B57-4* 1.37 ± 0.085 0.2116

A-101 B59-3 1.32 ± 0.141 0.2243

W29 ura3-302* 1.29 ± 0.306 0.2371

*: with uracil (2%).

Table 4 Bioscreen C growth of selected Suc+ Y. lipolytica strains in MMT (pH 7.2) with sucrose (1%) and minimal peptone doses. Only positives results were shown.

Y. lipolytica strain

Peptone (%)

Biomass ODmax

µmax (h-1)

Lag phase duration (h-1)

A-101 B56-5

0.005

1.61 ± 0.029 0.158 5

A-101 B54-6* 1.46 ± 0.058 0.159 20

A-101 B57-4* 0.96 ± 0.115 0.135 LN

A-101 A18* 0.41 ± 0.023 0.167 LN

W29 ura3-302* 0.94 ± 0.132 0.151 LN

A-101 B56-5

0.0025

1.3 ± 0.159 0.134 8

A-101 B54-6* 1.54 ± 0.058 0.081 22

A-101 B57-4* 0.48 ± 0.020 0.135 LN

W29 ura3-302* 0.81 ± 0.110 0.207 16

A-101 B54-6* 0.001

0.93 ± 0.167 0.113 28

W29 ura3-302* 0.43 ± 0.137 0.155 20

A-101 B54-6* 0 0.59 ± 0.219 0.149 24

*: with uracil (2%); LN: linear growth.

medium and strain dosage as well as during the

cultivation, the shaking regime and temperature

maintenance and finally a 15 minutes cycling of

turbidity (OD) measurements. Single Bioscreen C

study provides a vast amount of easily comparable

data about microorganisms growth. According to

Begot et al. [19], the obtained data were similar to

bioreactor ones (identical generation time). The

repeatability of OD values obtained with Bioscreen C

was high. Robak [16] has proved that the maximal

standard deviation of OD measure was 15%. In the

same study, a competition between acetate and ethanol

consumption by Y. lipolytica strains, as well as

between acetate and glycerol, was demonstrated for

the first time.

Production and secretion of invertase allows Y.

lipolytica yeast to growth on sucrose as a sole carbon

source. Invertase encoded by suc2 gene from S.

cerevisiae under the control of PXPR2 from Y. lipolytica

was effectively expressed in all transformants of Y.

lipolytica A-101. MMT medium has a well defined

composition, therefore, the perturbances caused by YE

(yeasts extracts) utilization were avoided. YE used by

others falsify the results of PXPR2 induction by peptone,

because Merck YE consists in 78% of peptone.

Forster et al. [7] have studied peptone addition in the

concentration of 0.1-5 g/L and have obtained the

maximal invertase expression for Y. lipolytica

H222-S4 T5 when 2 g/L (0.2%) of peptone were

added to the medium. They noted no effect with

further increase of peptone concentration. Peptone

concentration of 1.7-2.5 g/L (0.17%-0.25%), as

inductor of PXPR2, was also analyzed by Nicaud et al.

[2] and Chang et al. [3]. Results obtained in this study

clearly demonstrate that for induction of Y. lipolytica

A-101 Suc+ transformants growth smaller amount of

peptone is needed. Peptone added to the MMT

medium in the concentration range from 0.0025% to

0.01% has the same effect on strains growth on

sucrose. Only the diminution of peptone to a very

small concentration (0.001%) led to weaker growth

(1.6-1.8 times) and prolonged lag phase (2 times).

This has to be understood as a smaller promoter

induction. In conclusion, for transformants of Y.


1106

lipolytica A-101 the appropriated peptone dose in the

medium has to be 0.0025% (0.025 g/L). This dose is

even 100 times smaller than the one recommended by

others. Recently, Moeller et al. [20] confirmed that

only residual peptone concentration is sufficient for

PXPR2 induction. In studies of Lazar et al. [18], the

amount of peptone added as YE (0.3 g/L) was

sufficient for promoter induction. Concentration of 0.3

g/L of YE is equivalent to 0.234 g/L (0.023%) of

peptone. Optimization of medium composition,

especially concerning inductor dose of gene encoding

biotechnologically important protein expression, is an

economic necessity. The determination of minimal

peptone dose for PXPR2 allows a significant benefit in

medium costs.

Tested in that study Suc+ transformants growth on

sucrose (considered as increase of OD) was optimal in

pH 7.2. This pH value is higher than the optimal one

reported by others but the author have to notice that

Bioscreen C study was carried out in MMT medium

without any pH regulation. Because of the secretion of

citric acid, the pH rapidly decreased in the samples

and the growth in the medium initially controlled at

pH 7.2 was higher than in the medium of pH 6.8. In an

earlier publication, the author have demonstrated that

in bioreactors studies with pH control, the optimal pH

for induction of PXPR2 in some of here tested strains

was 6.8 [18]. The same optimal pH value for promoter

induction was reported by Forster et al. [7] for Y.

lipolytica H222-S4 invertase and by Chang et al. [3]

for α-amylase expression under the PXPR2. This pH is

exactly the same as found by Barth and Gaillardin [11]

for optimal expression of alkaline protease, naturally

controlled by PXPR2.

The level of sucrose uptake by Suc+ clones Y.

lipolytica A-101 was different in function of pH and

peptone concentration. In optimal induction condition

(pH 7.2 and 0.01% of peptone in MMT medium) the

best growing strains consumed 79%-86% of sucrose.

Bioscreen C studies did not allow the author to

determine the order of assimilated monosaccharides,

the author demonstrated that the majority of clones

Suc+ of Y. lipolytica A-101 firstly consumed glucose [8].

However, the rates of sucrose hydrolysis and uptake

of glucose and fructose were different [18].

Integration of suc2 gene under PXPR2 into genome of

Y. lipolytica strains led to different gene expression

level (growth on sucrose), despite an identical cassette

used for transformation. Even clones obtained by

homologous integration did not show similar growth

and consumption of sucrose. Three of Y. lipolytica

clones (A-101 B56-5; A-101 B54-6 and W29

ura3-302) showed similar and very important growth,

however, they differed in suc2 copy number and in the

gene integration site. The first one has two copies

integrated tandemly but not in ura3 sequence [18].

The second and third ones have one copy integrated in

ura3 [2, 8]. Therefore, the site of integration is

important for PXPR2 regulation and invertase secretion.

The author could presume both (copies number and

site of cassette integration into genome) influence the

way of promoter induction and gene expression. It

will be interesting to study the gene fragment

upstream of tandemly integrated suc2 sequences,

which also contain fragment of ura3d. Recently,

Blazek et al. [21] described the outcomes of the study

on UAS1B region of PXPR2 as enhancer of TEF

promoter conducting expression of reporter genes.

They revealed that introduction of 16-32 copies of

UASIB led to 400 folds increase in gene expression

measured by hGFP synthesis. Gasmi et al. [22]

investigated the effect of the presence of CACA

sequence at translation initiation site on expression of

human 2b interferon in Y. lipolytica. Tirosh et al. [23]

discussed promoter architecture and gene expression,

claiming that gene expression regulation is the main

difference between the two closely related yeasts

species. Genes whose promoter contains a TATA box,

showed high expression divergence which compared

with TATA-less genes. TATA-containing genes were

also highly variable between natural isolates and

experimentally evolved yeast strains. Evolution of


1107

gene expression regulation in yeasts is still an

intensively studied field [25-28].

In summary, the authors’ results demonstrated that

the optimal pH (7.2) was similar to the value (6.8)

recommended by others. Contrary to the minimal

concentration of peptone required for the growth of Y.

lipolytica A-101 Suc+ transformants in MMT on

sucrose, which is only 0.0025%. On the base of the

authors’ results, in the designing of large scale

processes involving PXPR2 controlling heterologous

gene expression in Y. lipolytica the peptone

concentration regarded as optimal inductor dose has to

be 100 times smaller than recommended so far. It is an

evident gain on the medium cost.

Acknowledgments

Authors would like to thank Prof. Gerold Barth and

Dr. Stephan Mauersberger from Institute of Microbiology,

University of Technology, Dresden, Germany, for

plasmids and help in yeast transformation.

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In Vitro Study on Virulence Potentials of Burkholderia

pseudomallei Isolated from Immunocompromised

Patients

Hadeel Tawfiq Al-Hadithi Rana and Muhammad Abdulnabi

Dept. of Biology, College of Science, University of Basrah, Basrah, Iraq

Received: April 10, 2012 / Accepted: May 31, 2012 / Published: October 30, 2012.

Abstract: Eighty four throat swabs were obtained from Basrah General Hospital inpatients (N = 34): 17 were suffering from renal

failure and the other 17 were diabetics; and from outpatients (N = 50). Throat swabs were cultured first in the selective media

Ashdown’s broth then subcultured on Ashdown’s agar to isolate Burkholderia pseudomallei which was recovered from seven cases

(8.33%). Four isolates were from renal failure patients (23.53%), two from diabetic patients (11.76%) and the seventh isolate was

from an outpatient with tonsillitis. All isolates were able to produce capsules, form filament chains, exhibit swarming motility and

were arabinose non assimilators (Ara-) indicative of their virulence. Additionally, isolated B. pseudomallei were found to produce

protease, lipase, hemolysin, and lecithinase and were able to produce biofilm, the root of many troublesome persistent infections that

resist antibiotic treatment. Susceptibility of the seven isolates of B. pseudomallei toward 11 antibiotics was assessed, isolates were

found multiply resistant to all antibiotics apart from ciproflaxin. This study confirms for the first time isolation of B. pseudomallei

from immunocompromised patients in Basrah city of Iraq and describes their virulence potentials.

Key words: B. pseudomallei, virulence potentials, biofilm, antibiotic susceptibility, immunocompromised patients.

1. Introduction

B. pseudomallei are Gram- negative bipolar aerobic

motile rod-shaped bacteria. It is a soil saprophyte

endemic to many areas in Southeast Asia [1, 2] and

northern Australia but is sporadically isolated in

subtropical and temperate countries [3, 4]. Although it

is very rarely seen in patients in the United States,

O’Sullivan et al. reported pulmonary B. pseudomallei

infection in a young girl with cystic fibrosis who had

never traveled to Asia or Australia [5].

B. pseudomallei are present in stagnant water,

paddy fields and infection is via the skin through

Corresponding author: Hadeel Tawfiq Al-Hadithi Rana,

Ph.D., professor, research field: microbiology. E-mail: [email protected].

abrasions or aspiration or ingestion of contaminated

water and inhalation of dust from contaminated soil

[6-8]. It is a human and animal pathogen which causes

melioidosis [9-11], a little known disease that is

endemic, and is easily be mistaken for tuberculosis or

a fungal infection [12, 13]. Risk factors for subjective

infection with B. pseudomallei include: diabetes, renal

failure, chronic lung disease, thalassaemia, iron

overload, cystic fibrosis, influenza A, carcinoma,

radiation therapy, pregnancy and immunosuppressive

drugs [14-18]. Besides, it might cause bacteraemic

pneumonia in a diabetic patient [19].

Moreover, Estivals et al. reported that septicemia,

bilateral community acquired pneumonia and

empyema could result due to B. pseudomallei with a

In Vitro Study on Virulence Potentials of Burkholderia pseudomallei Isolated from Immunocompromised Patients

1110

favorable outcome following prolonged specific

antibiotic therapy [20].

Ulett et al. indicated that virulence of selected B.

pseudomallei isolates is variable and the level of

virulence is important in disease pathogenesis and

progression [21]. Moreover, Tumapa et al. study

confirmed the utility of a range of approaches in

defining the presence and significance of genomic

variation in natural populations of B. pseudomallei

and indicated that horizontal gene transfer contributes

to the genetic diversity of this pathogen and may be an

important determinant of virulence potential [22].

Hence, the first aim of the present study is to assess

whether B. pseudomallei could be recovered from

inpatients suffering from renal failure and diabetes in

addition to outpatients attending Basrah General

Hospital and secondly, to study their virulence

capabilities.


2.1 Samples

Eighty four throat swabs were obtained from

inpatients (N = 34) including those with renal failure

(N = 17) and diabetes (N = 17), in addition to 50

throat samples from outpatients suffering from

tonsillitis. All samples were from inpatients and

outpatients attending Basrah General Hospital, Iraq.

2.2 Isolation and Identification

Swabs were cultured out in Ashdown’s broth which

consists of: 15 g Trypton soy peptone, 4% glycerol, 5

mg of crystal violet per liter, 50 mg of neutral red per

liter, pH was adjusted to 7-7.4 sterilized in autoclave

then cooled to about 40 °C and 20 mg of colistin per

liter was added aseptically, then incubated at 35 °C [23].

When broth turned red after 5-7 days, loopfulls were

subcultured on Ashdown’s selective agar plates [24].

Wrinkled purple grown colonies giving rise to a

metallic sheen with earthy smell were subjected to

Gram stain. Identification of B. pseudomallei was

confirmed by subjecting colonies demonstrating Gram

negative, bipolar staining rods to the following

characterization tests: production of catalase, oxidase

and gelatinase, oxidation/fermentation test, oxidation

of lactose and maltose, growth at 37 °C and 42 °C,

hydrolysis of arginine and decarboxylation of lysine

and orthinine [25-28].

2.3 Determination of Virulence Factors Of Isolated B.

pseudomallei

2.3.1 Formation of Filamentous Chains

Isolates were grown on nutrient agar plates for

18-48 h at 35 °C. Smears were prepared and stained

with methylene blue then examined microscopically

for the detection of filamentous chains [29].

2.3.2 Formation of Capsules

Negative staining using Indian ink was performed

to examine the production of capsules by isolates

grown for 48 h at 35 °C [27].

2.3.3 Swarming Motility

Loopfuls of normal saline (NaCl 0.85%) containing

grown isolates at concentration of 104 were placed

centrally on plates containing semisolid nutrient

medium (0.5 g agar, 1.3 g nutrient broth in 100 mL

distilled water). Plates were incubated at 30 °C for

16-18 h [30]. To evaluate ability of isolates for

swarming motility, diameter of growth was measured

by millimeter after incubation.

2.4 Determination of Biotypes

Each isolate was tested for its ability to assimilate

L-arabinose which was determined according to

Wuthiekanun et al. [28]. Loopfulls of 24 h grown

cultures at 37 °C were spotted on arabinose agar

medium which consists of: 75 mL of agar solution

(2%), 2 mL of L-arabinose solution (10%) as the only

source of carbon, 25 mL of minimal salt solution

consisting of: 5 g NaCl, 0.2 g MgSO4, 10 g K2HPO4, 1

g KH2PO4, 1 g (NH4)2SO4, 0.05 g Fe SO4 dissolved in

liter distilled water [31]. Controls were prepared by

culturing isolates on the same medium with the

addition of glucose instead of L-arabinose. After


1111

incubation at 35 °C for 48 h, grown isolates were

reported as arabinose assimilators (Arb+ biotype).

2.5 Enzyme Production

Ability of the seven isolates to produce the

following enzymes was determined: protease [32],

haemolysin [33], lipase and lecithinase [27].

2.6 Detection of Biofilm Production

Isolates were inoculated into 2 mL of tryptone soy

yeast broth (TSYB) and incubated at 37 °C for 48 h,

0.1 mL of each culture was suspended into tubes

containing 10 mL of TSYB to obtain a dilution of

1:100 [30]. 3 mL of each suspension was transferred

into small tubes (75 × 10) and were lightly covered

with foil and incubated at 37 °C for up to 30 h. For

detection and quantification of biofilm formation, the

tubes were rinsed thoroughly and vigorously with

water and the remaining cells were stained with 0.5%

crystal violet solution for 15 min at room temperature.

The crystal violet-stained biofilm was then solubilized

by the addition of 6 mL of 100% ethanol for 10 min at

room temperature and optical density (OD570) of 200

μL of the resultant suspension was measured by a

spectrophotometer. Replicates of three tubes per

isolate were examined for each experiment.

Uninoculated broth control was included in each

experiment.

2.7 Antibiotic Susceptibility Test

Antibiotic susceptibility was investigated by disc

plate method [35] using Muller Hinton agar towards

11 antibiotics. These are: ampicillin (10 μg),

trimethoprim sulpha methoxazole (25 μg), tetracycline

(30 µg), tobramycin (10 µg), gentamycin (10 µg),

amoxcillin clavulanic acid (30 µg), chloramphenicol

(30 µg), cephatollin (30 µg), erythromycin (15 µg),

cefotaxime (30 µg) and Ciprofloxacin (5 µg).

3. Results

Gram stained smears prepared from wrinkled purple

grown colonies with metallic sheen and earthy smell

demonstrated that Gram negative short biopolar

staining rods were preliminary identified as B.

pseudomallei (Fig. 1).

These rods were confirmed as being B.

pseudomallei when they are motile, oxidative, positive

to catalase, oxidase and produce gelatinase, oxidize

maltose, grow at 37 °C and 42 °C, hydrolyse arginine,

and did not decarboxylate lysine or orthinine.

Table 1 illustrates percentage recovery of B.

pseudomallei from inpatients and outpatients

attending Basrah General Hospital, Iraq. Out of 84

throat swabs, B. pseudomallei was recovered from 7

patients (8.33%). Four patients suffering from renal

failure have demonstrated the highest percentage

recovery (23.52%) of B. pseudomallei followed by

diabetics (11.76%).

Table 2 describes status of patients from which B.

pseudomallie was isolated. Age of patients ranged

from 19 to 69 years, similar recovery rate of B.

pseudomallie was recorded in both sexes.

Fig. 1 A) Characteristic purple colored colonies of B. pseudomallei with metallic sheen grown on Ashdown selective agar. B) Bipolar staining rods.

A B


1112

Table 1 Percentage recovery of B. pseudomallie from clinical sources.

Throat swabs N n (%)

Patients with renal failure 17 4 (23.52)

Patients with diabetes 17 2 (11.76)

Outpatients 50 1 (2)

Table 2 Description of cases from which B. pseudomallei was isolated.

Gender Age/Yrs. Patients

Male 60 1—A Diabetic patient (ICU)

Female 53 2—A Diabetic patient (Medicine Ward)

Male 34 3—A Patient with Chronic Renal Failure & Hypertension (Dialysis unit)

Female 45 4—A Patient with Renal Failure (Dialysis Unit)

Female 20 5—A Patient with Renal Failure (Dialysis Unit)

Male 69 6—A Patient with Renal Failure (Dialysis Unit)

Female 19 7—An Outpatient with Tonsillitis

Fig. 2 A) Swarming motility; B) Filamentous chains; C) capsules.

The seven isolates of B. pseudomallie

demonstrated swarming motility, a pseudopodial-like

movement of packs of cells, form filamentous chains

and capsules (Fig. 2).

Also, isolates were found to be proteolytic,

lipolytic, hemolytic and lyse lecithine, apart from

one isolate, was not capable of lysing lecithine or

R.B.Cs (Table 3). The seven isolates did not

assimilate arabinose.

Four isolates selected to examine their ability to

form biofilm were successful in forming biofilms

(Fig. 3).

Almost similar optical density ranging from

1.847-1.864 was recorded by isolates of B.

pseudomallei under study (Table 4).

Isolates of B. pseudomallei demonstrated multiple

A B

C


1113

Table 3 Enzymes produced by the seven Burkholderia pseudomallei isolates.

Source of Isolate

Enzymes Lipase Lecithinase Hemolysin Protease

R1* + + +sß +

R2* + + +sß +

R3* + + +sß +

R4* + + +sß +

D1** + + +sß +

D2** + - - +

T1*** + + sα+ +

100% 85.71% 85.71% 100%

* R1, R2, R3 and R4: Isolates from patients with renal failure; ** D1 and D2: Isolates from diabetic patients; *** T1: Isolate from one outpatient with tonsillitis.

resistances toward 10 antibiotics under study but were

susceptible to ciproflaxin only.

4. Discussion

Culturing of B. pseudomallei on selective medium

remains the gold standard for the diagnosis of

melioidosis [36]. In the present study, out of 34

inpatients, four were suffering from renal failure

(11.76%) and two were diabetics (5.88%), in addition

to one (2%) out of 50 outpatients had B.

pseudomallei. Isolates were obtained from throat

swabs cultured on Ashdown’s agar after being

cultured first in Ashdown’s broth [23] which allows

bacteria to flourish and provides consistent results,

although direct plating on Ashdown’s medium was

adopted by Wuthiekanun et al. [37]. Nevertheless,

Cheng et al. reported that isolation relies on culture

and the growth of even a single colony of B.

pseudomallei from anybody site is indicative of

disease [38].

Although Chanchamroen et al. [39] indicated that

diabetes mellitus is the major predisposing factor for

melioidosis but the present study found that percentage

recovery of B. pseudomallei from inpatients suffering

from renal failure was as twice as those of diabetics.

Strains of B. pseudomallei were identified

phenotipically according to the criteria described for

Gram-negative nonfermentative bacilli and

by characteristics of colonies on Ashdown’s selective

Fig. 3 Filamentous chains, capsules and biofilm formation (aggregated cells) by B. pseudomallei (crystal violet stain).

Table 4 Optical density of biofilms formed by four clinical isolates of B. pseudomallei. Source of isolate Optical density

R1* 1.847

R2* 1.854

D1** 1.860

T1*** 1.864

* R1 and R2 Isolates from patients with renal failure; ** D1: An isolates from a diabetic patient; *** T1: An isolate from outpatient with tonsillitis.


1114

medium [24, 40, 41]. Bipolar staining rods were

demonstrated remarkably, probably because of

intracellular deposits of b-hydroxy butyric acid [42, 43].

Also, motility was evident which verify possession of

flagellum, the important and necessary virulence

determinant of B.pseudomallei during intranasal and

intraperitoneal infection of mice [44]. Besides,

Tunpiboonsak et al. [45] confirmed that motility of

this intracellular pathogen is generally crucial for their

survival in a natural environment and for systemic

infection inside a host.

Despite similarity in morphologies and

antigenicities, B. pseudomallei was reported to have

two distinctive stable biotypes: the

arabinose-assimilators (Ara+ biotype) which are

non-virulent and the non arabinose-assimilators (Ara-

biotype) which are virulent in experimental animals

[46, 47]. In the present study, the seven isolates did

not assimilate arabinose which is indicative of their

virulence [48]. This result validates the findings of

Thepati et al. [47] who reported that Ara- biotype is

found in almost all B. pseudomallei clinical isolates.

Reckseidler-Zenteno et al. reported that disease

caused by B. pseudomallei causes high relapse rate

(when compared to other bacterial infections), and

suggested that it is due to the reactivation of the

biofilm forming bacteria which also provide resistance

to antimicrobial agents [49]. In the present study, high

density biofilm forming capacity was demonstrated by

isolated B. pseudomallei, using glass tube assay model

which gave consistent results, in addition to the

resistance of isolates to 10 out of 11 antibiotics

examined, confirm virulence of these isolates.. This

verifies the report of Sawasdidoln et al. that B.

pseudomallei is inherently resistant to a number of

antibiotics, and even with aggressive antibiotic

therapy, the mortality rate remains high [50]. However,

Donlan [51] and Pibalpakdee et al. [52] proved that

biofilm-forming capacity is at the root of many

troublesome persistent infections that resist antibiotic

treatment. However, the mechanism on how biofilm

can provide tolerance to antimicrobials is still unclear.

Hence, resistance of this organism to a number of

antibiotics has created a need for the development of

other therapeutic strategies, including the

identification of novel therapeutic goals.

5. Conclusion

The present study confirms for the first time, the

presence of B. pseudomallei in Basrah city. It sheds

light on their characteristics and virulence potentials

to increase awareness among microbiologists in the

region to recognize subsequent cases even when there

is no clinical suspicion. Moreover, biofilm formation

and multiple resistance to antibiotics demonstrated in

the present study emphasize the need for prolonged

eradication therapy and regular clinical and

microbiological monitoring.

Acknowledgments

Appreciation is due to the management of Basrah

General Hospital for facilitating collection of throat

swabs.

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[41] A.L. Walsh, V. Wuthiekanun, The laboratory diagnosis of melioidosis, British Journal of Biomedical Science 53 (1996) 249-253.

[42] T.J.J. Inglis, B.J. Mee, B.J. Chang, The environmental microbiology of melioidosis, Review of Medicinal Microbiology 12 (2001) 13-20.

[43] L.D. Sprague, H. Neubauer, Melioidosis in animals: A

rewiew on epizootiology, diagnosis and clinical presentation, Journal of Veterinary Medicine 51 (2004) 305-320.

[44] K.L. Chua, Y.Y. Chan, Y.H. Gan, Flagella are virulence determinants of Burkholderia pseudomallei, Infection and Immunity 71 (4) (2003) 1622-1629.

[45] S. Tunpiboonsak, R. Mongkolrob, K. Kitudomsub, P. Thanwatanaying, W. Kiettipirodom, Y. Tungboontina, et al., Role of a Burkholderia pseudomallei polyphosphate kinase inan oxidative stress response, motilities and biofilm formation, Journal of Microbiology 48 (1) (2010) 63-70.

[46] M.D. Smith, B.J. Angus, V. Wuthiekanun , N.J. White, Arabinose assimilation defines a nonvirulent biotype of Burkholderia pseudomallei, Infection and Immunity 65 (10) (1997) 4319-4321.

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[50] C. Sawasdidoln, S. Taweechaisupapong, R.W. Sermswan, U. Tattawasart, S. Tungpradabkul, S. Wongratanacheewin, Growing Burkholderia pseudomallei in biofilm stimulating conditions significantly induces antimicrobial resistance, Public Library of Science 5 (2) (2010) e9196.

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The Control of Malaria among PLWHA in Calabar, Cross

River State, Nigeria

Patience Edoho Samson-Akpan, Olaide Bamidele Edet, Ekaette Francis Asuquo, Mary Achi Mgbekem and Idang

Neji Ojong

Department of Nursing Science, University of Calabar, Calabar, Cross River State 54001, Nigeria

Received: February 29, 2012 / Accepted: June 22, 2012 / Published: October 30, 2012.

Abstract: The purpose of the study was to examine RBM programme’s efforts at controlling malaria among PLWHA and explore their perception of the control strategies. The study was a descriptive survey involving guided interviews of top managers of Roll Back Malaria (RBM) programme. A structured questionnaire was administered to 108 PLWHA attending an HIV/AIDS clinic in a secondary health facility in Calabar. Data were analyzed using descriptive statistics. Thematic analysis revealed that RBM programme strategies include effective case management, promotion of Long Lasting Insecticide Treated Nets (LLINs), intermittent preventive treatment (IPT) and integrated vector management (IVM). Complementary results showed that 104 (92%) admitted accessibility to malarial treatment. Although 83 (57.7%) of PLWHA have LLINs, only 63 (42.3%) use them. Majority of the respondents 89 (60%) have not heard of indoor/outdoor residual spraying (IRS). How to get IRS services and lack of money to buy it were listed as a barrier to its use. Malarial treatment was accessible to PLWHA. The barriers to the use of ITN and IRS could be addressed through free distribution of odorless ITN and IRS to PLWHA. Higher rates of utilization of the products can be achieved through behavioural change communication. Key words: Malaria control, PLWHA, Roll Back Malaria Programme, Nigeria.

1. Introduction

Malaria and HIV are among the two most important

global health problems of our time. Malaria in

combination with HIV cause more than 4 million

deaths a year and are both concentrated primarily in

Sub-Saharan Africa of which Nigeria is located, Asia

and South America. Furthermore, more than 500

million cases of malaria occur every year and at least a

million of them cause deaths. An estimated 30-36

million people are living with HIV in Africa, resulting

in more than 3 million deaths [1].

Although early studies failed to demonstrate

significant interaction, now there is a good evidence

for a dual interaction between HIV/AIDS and malaria

[2]. Studies in 2006 highlighted the interaction

Corresponding author: Patience Edoho Samson-Akpan,

Ph.D., senior lecturer, research field: health promotion. E-mail: [email protected].

between malaria and HIV infection, that malaria might

be fuelling the spread of HIV in areas of Sub-Saharan

Africa including Nigeria while HIV may be playing a

role in boosting adult malaria infection rates. Based on

a study in a Kenyan city with high levels of both

malaria and HIV, researchers calculated that the

interaction increased AIDS cases by 8% and malaria

by 13% [1]. This result is affirmed by other reports

that once malaria gets into the blood of a person living

with HIV, it increases the level of HIV by up to 10

times during a malarial episode. This significantly

increases the risk of them infecting a sexual partner

[3].

Some of the effects of malaria and HIV interaction

in HIV positive adults reveal that PLWHA are

especially vulnerable to malaria and will suffer more

often and severely from malaria once their immune

system starts declining. HIV not only increases the

The Control of Malaria among PLWHA in Calabar, Cross River State, Nigeria

1118

incidence and severity of malaria, it also

compromises malaria treatment by decreasing the

response to standard anti-malaria treatment. For HIV

positive adults with a weakened immune system (a

low CD4 Count), anti-malarial drugs are less likely

to be effective. Lastly, malaria contributes to increase

viral load among HIV-positive people which can

potentially accelerate the progression from HIV to

AIDS [1]. These interactions make PLWHA to be

double vulnerable to increased morbidity and

mortality.

Previously malaria control had focused on case

management of malaria with chloroquine as the

traditional drug of choice. However, the increase in

chloroquine resistant to malaria necessitated

additional method of malaria control [4]. Therefore,

in 2000, Roll Back Malaria (RBM) Programme was

launched in Nigeria as a WHO initiative which is a

global strategy to improve health systems with the

goal of a 50% reduction in malaria death by 2010 [5, 6].

A key Millennium Development Goal (MDG) is to

halve malaria and AIDS-associated mortality by

2015 and almost other MDGs are related to

achieving success in reducing malaria and HIV

burdens [7].

The Nigerian Government has been collaborating

with a number of international organizations including

World Bank, WHO, UNDP, UNICEF on a campaign

tagged “Roll Back Malaria” [5, 7]. This effort has led

to the establishment of National Malaria Control

Programme (NMCP) that seeks to unify all of the

disparate pieces of the Nigerian malaria control

strategy at national, regional and local government

levels. Through RBM Programme, malaria control

comprises of four primary strategies namely: case

management using artemisinin-based combination

therapies; Long Lasting Insecticide-Treated Nets

(LLINs), and other vector control measures; providing

malaria treatment and intermittent preventive

treatment for pregnant women; improving malaria

epidemic preparedness and response.

Regarding the use of LLINs, it is estimated to be

twice as effective as untreated net and offer 70% of

protection when compared to no net. LLINs have been

found to reduce clinical malaria by over 50% [8]. The

established efficacy of ITNs in malaria prevention

made the African Head of States at the Abuja summit

in 2000 to set a target of 60% coverage by 2005 for

pregnant women and children < 5 years. The target

was subsequently raised in 2010 to 80% by WHO in

2005 [9]. However, accesses to nets has remained

poor across many African countries [10]. Studies have

equally shown that the awareness in Nigeria about

LLINs is high [11, 12]. However, these studies have

equally revealed that LLINs use at night prior to the

studies was below 37%. Some of the challenges in

using ITNs identified in the literature were high cost

of the net, poor perception of the chemical used as

being dangerous, low utilization of ante natal care

among others [13, 14].

Another global strategy to reduce malaria burden is

the use of Indoor Residual Spray (IRS) which remains

the most widely used malaria vector control method

[7]. The main effect of IRS is to kill off mosquitoes

entering the houses and resting on sprayed surfaces.

IRS is a method for community protection and to

achieve its full effect, IRS requires a high level of

coverage, in space and time, of all surfaces where the

vector is likely to rest, with an effective dose

insecticide. IRS requires the acceptance of all

population to spray once or twice a year and a

reasonable preservation of sprayed surfaces without

re-plastering in contrast with ITNs [7].

Malaria as an endemic disease in Nigeria poses a

major challenge to the country as it impedes

economic development. Malaria is responsible for

the huge economic loss of about 132 billion naira

(US$880 million) annually due to cost of treatment,

loss of man hour and school absenteeism and other

indirect cost [7, 15]. Malaria in combination with

HIV/AIDS increases morbidity and mortality

including economic cost. In view of the government


1119

strategy to control malaria using RBM programme,

there is no study to assess the RBM programme

efforts at controlling malaria among People Living

with HIV/AIDS (PLWHA) and their perception of

the control strategy. The study was therefore

undertaken with the following objectives:

(1) To assess Cross River State RBM programme

with regards to malaria control among PLWHA;

(2) To ascertain the proportion of PLWHA who

have access to and are using LLINs in the chosen

public hospital Calabar;

(3) To identify the challenges in accessing ITNs by

PLWHA in the chosen public hospital;

(4) To ascertain the opinion of PLWHA on

accessibility to anti-malarial drugs in the chosen

hospital;

(5) To determine the proportion of PLWHA in the

chosen public hospital who have had their houses

treated with IRS;

(6) To identify the problems encountered by

PLWHA accessing IRS in the chosen hospital.

2. Methods and Materials

The study was carried out in Calabar which is

located in the mangrove swamp forest belt in the

south-south part of Nigeria. Calabar is the capital of

Cross River State and it is fast developing into a

tourist attraction centre. The study sites were the

biggest state owned hospital which has a donor

assisted unit for PLWHA and a RBM office both of

which are situated in Calabar.

The study was a descriptive survey which utilized

an incidental sample of 149 PLWHA who were

attending out-patients clinic of the selected hospital

between November 24 and December 5, 2008. Two

top managers of Cross River State RBM programme

were also involved in the study.

The instruments for data collection were a self

developed and a well validated questionnaire and an

interview guide. The questionnaire was pretested

using PLWHA in another secondary health facility not

used in the study. The reliability co-efficient of the

instrument was 0.87 which was considered good

enough to be used for the study. The interview guide

was also assessed by two experts in the Departments

of Public Health and Test and Measurement of the

University of Calabar, Nigeria.

Data collection was done through three research

assistants who work in the Heart to Heart Units of the

hospital. This unit only attends to PLWHA. The

research assistants were trained by the researchers on

the essence of the research and how to collect data

from respondents. Prior to the survey, the purpose of

the study was explained to the respondents and their

consent sought. Participants were ensured anonymity

and confidentiality of the information which was to be

used for research purposes. Permission to carry out the

research was also obtained from the Ethical

Committee of the hospital. Descriptive statistics was

used to analyze the data. Results were presented in

simple frequencies and percentages.


3.1 Socio-Demographic Characteristics of the

Respondents

Table 1 shows the socio-demographic

characteristics of the respondents. The majority of the

respondents were females 90 (60.4%); mostly between

ages 21-30 years 62 (41.6%). Marital status revealed

that the majority were single 69 (46.3%) and mostly

of Christian origin 138 (92.6%).

Regarding ethnicity, the majority were the Efiks

58 (38.9%) followed by the Ibibios 49 (32.9%). Most

the respondents had no formal education 64 (43%)

and no source of income 66 (44.3%). Most of the

respondents 37 (24.8%) were unemployed and

trading 31 (20.8%), while 28 (18.8%) were involved

in business.

3.2 Assessment of Cross River State RBM Programme

with Regards to Malaria Control among PLWHA

The results came in form of direct reporting from


1120

Table 1 Socio-demographic characteristics of the respondents on the control of malaria among PLWHA in Calabar, Nigeria, 2008.

Characteristics of the respondents Frequency Sex

Male 59 (39.6%) Female 90 (60.4%)

Age of respondents in years 11-20 5 (3.4%) 21-30 62 (41.6%) 31-40 52 (34.9%) 41-50 18 (12.1%) 50+ 12 (8.1%)

Marital status Single 69 (46.3%) Married 67 (45.0%) Separated/divorced/widowed 13 (8.7%)

Religion Christianity 138 (92.6%) Islam 9 (6.0%) Others 2 (1.4%)

Ethnicity Efik 58 (38.9%) Ibibio 49 (32.9%) Ekoi 13 (8.7%) Others 29 (19.5%)

Highest educational qualification No formal education 64 (43%) First School Leaving Certificate 23 (15.4%) West African School Certificate 37 (24.8%) Diploma 14 (9.4%) Degree 11 (7.4%)

Income level No income 66 (44.3%) < 0,000 31 (20.8%) < 20,000 23 (15.4%) < 40,000 15 (10.1%) > 41,000 14 (9.4%)

Occupation Civil service 29 (19.5%) Farming 22 (14.8%) Trading 31 (20.8%) Business 28 (18.8%) pensioners 2 (1.3%) Not employed 37 (24.8%)

two top managers of the RBM programme in Cross

River State. Malaria control at the national level is

stepped down to the state level with major partners:

WHO, UNICEF, UNDP, World Bank, Canadian Red

Cross Society, USAID among others involved in the

exercise.

In Cross River State there are many NGOs such as

Africare, Interface Health Care and Society for Family

Health including 125 civil societies collaborating with

the state RBM programme to control malaria. Africare

concentrates on health integrated programme focusing

on Biase, Obubra, and Abi LGAs working on behalf

of Shell. Interface Health produces insecticide treated

nets for the RBM programme. Society for Family

Health collaborates with the state RBM programme to

build capacity of health care workers, patent medicine

store vendors, pharmacist, and role mothers on the

control of malaria.

The state is a global funded state for HIV,

tuberculosis and malaria. The state RBM Programme

also collaborates with the Institute for Tropical

Diseases Research and Prevention of the University of

Calabar to carry out operational research to test the

efficacy of drugs and control activities. It was reported

that 15% of the annual budget on health is supposed to

be apportioned for malarial control.

Currently, everybody is a stakeholder in the control

of malaria. The private sector made up of pharmacies,

private clinics, patent medicine stores, among others

control more than 43% of the cases. The state has the

following structures on ground for effective control of

malaria: appointment of the state malaria programme

manager with his team in its Headquarters in Calabar,

18 focal persons were also appointed for the 18 local

government areas. These appointees are responsible

for advocacy, sensitization; distribution of

commodities to designated health facilities and

evaluation of malaria control activities. Evaluation

meetings are held once every month.

One of the main strategies for the control of malaria

includes effective management of all persons at risk

including the vulnerable groups such as children under

5 years old, pregnant mothers and PLWHA. Effective

case management involves prompt and appropriate

treatment of patient within 24 hours of illness using

the right artemisinin-based combination therapies.

Intermittent Preventive Treatment during pregnancy

(IPTp) is used as a preventive strategy for pregnant

women. Pregnant women who live positively are

given three doses of sulfadoxine-pyrimethamine (SP)


1121

during ante-natal period. The SP is free and accessible

in the hospital, health centres and other government

owned clinics. LLINs are given free of charge to

pregnant women who attend antenatal clinic at any

government health facility.

The non pregnant HIV positive people are educated

to sleep under the ITNs. The ITNs are sold at a

subsidized rate of one thousand naira (approximately

US$6.00) to PLWHA. Other preventive strategies

include multiple measures such as general health

education, promoting the use of long acting

insecticide treated nets (LLINs), integrated vector

management through indoor and outdoor residual

spray. Environmental management includes clearing

bushes, covering stagnant water, and burying cans.

Cross cutting areas involve behavioural change

communication using advocacy to stakeholders,

sensitization, and mobilization to create awareness on

the burden and methods of reducing the burdens of

malaria. Posters, handbills and jingles on radio are

used to create awareness.

The state RBM activities include monitoring and

evaluation which allows for accountability fostering

international and united dynamic partnership. These

reports on various activities of the state RBM

programme are in line with the global and national

objectives of RBM programme. The challenges

include poor funding which is less than 15% of the

annual budget on health and poor counterpart funding.

Other problems identified include poor health seeking

behavior of the people; lack of trained personnel

(focal persons at the local government level);

uncoordinated transfer of trained personnel;

uncommitted attitude of staff and poor utilization of

evidenced based decision; lack of transportation,

spraying equipments and information communication

equipments. Additionally, there is social apathy

towards malarial control messages (personal

communication with two top managers of Cross River

State RBM programme on November 27 and

December 4, 2008).

3.3 Proportion of PLWHA Who Have Access to and

Are Using LLINs and Challenges in Accessing LLINs

The results revealed that the majority of the

respondents 129 (86%) have heard about LLINs, 83

(55.7%) have LLINs and 63 (42.3%) were using

ITNs. The results of this study confirm other studies

which revealed that the awareness on LLINs was

high but the use of LLINs was however low [10-14].

Interaction between HIV and malaria makes

PLWHA to be more vulnerable therefore if they can

not access LLINs then they should be considered as

endangered species and Nigeria will remain under

developed because of the burdens of the two

infections.

The main reason for not using LLINs was lack of

money to buy the commodity 60 (40.27%); heat 11

(7.4%); some respondents did not know where to buy

the net 10 (6.7%). This is not surprising because

majority of the PLWHA are those with no formal

education and have no means of livelihood. Although

the LLINs are sold at a subsidized rate, it is still

impossible for them to buy the nets because of lack of

money.

3.4 Opinion of PLWHA on Accessibility to

Anti-Malarial Drugs in the Chosen Hospital

The results showed that the majority of the

respondents attested to anti malarial drugs being

accessible 71 (47.7%) and very accessible 66 (44.3%).

This result is not a surprise because most of the

PLWHA (63%) visit the selected health facility at least

once in a month to replenish their anti retroviral drugs.

Therefore, they use the opportunity to treat malaria if

they are affected.

3.5 Proportion of PLWHA Who Had Their Houses

Treated with IRS and the Problems Encountered by

PLWHA in Accessing IRS

The results highlighted that 89 (60%) respondents

have heard of IRS while only 55 (37%) have had


1122

their houses treated with IRS. This result was

expected because the unit in charge of IRS is not

situated in the hospital. Therefore, most of the

respondents after leaving the hospital premises may

not like to go and negotiate IRS services outside the

hospital. The major challenges to using IRS are lack

of money 60 (40.3%) and not knowing where to get

the services. The benefit of using IRS as a method

for community protection is enormous and this

protective method is recommended by WHO as RBM

preventive strategy. Therefore, the result of this study

is at variance with what is expected by WHO and

national RBM programmes [2].

4. Conclusion and Recommendations

Conclusively, the challenges encountered by the

RBM programme in Cross River State should be

promptly addressed. Malaria treatment was accessible

to participants, ITNs was moderately accessible while

IRS was not accessible to PLWHA. Based on these

findings, re-orientation of the staff of the RBM

programme to increase commitment and effectiveness

in the control of malaria is highly recommended. The

necessary facilities and equipment to enhance work

should be provided including adequate funding by the

government and counterpart partners. The political

will to control malaria should be provided by the

government. The barriers to the use of ITNs and IRS

should be removed through free distribution of ITNs

and increased accessibility to IRS at no cost to

PLWHA. Intensive behavioural change

communication should be carried out to increase

utilization of both ITNs and IRS.

Acknowledgments

The authors wish to acknowledge the

management of Cross River State Roll Back Malaria

Programme for granting us access to information on

malaria control measures in the state. The authors

also wish to acknowledge the contribution of Mrs.

Magdalene Nkang, nurses and caregivers of the

Heart to Heart Centre, General Hospital Calabar for

their assistance in the collection of data, and also

appreciate Mrs. Rita Etifit who was very

cooperative in the area of monitoring to ensure that

ethical standards of the institution’s Ethical

Committee were adhered to.

References

[1] Centre for Disease Control and Prevention Home Page,

Link between malaria and HIV, CDC, UNAIDS/WHO,

http://www.europeanallainceagainstmalaria.org/update/m

edia/malariaandhiv/en.pdf (accessed Sept. 8, 2011).

[2] WHO/RBM Home Page, Malaria and HIV interactions

and their implications for public health policy,

http://www.who.int/hiv/pub/prev_care/malaria/en/index.h

tml (accessed Sept. 8, 2011)

[3] United Nation International Children Emergency Fund

(UNICEF), 2003, Malaria and HIV, Technical Notes No.

6, 2003.

[4] A. Carrington, Malaria: Its human impacts, challenges

and control strategies in Nigeria [Online], 2001,

http://www.hcs.havard.edu/_epihccurrentissue/Fall2001/

Carring-ton.htm (accessed Nov. 3, 2010).

[5] C.A. Onoka, Public/private partnership in the

management and control of malaria, in: West African

College of Nursing Malaria in pregnancy: Implication for

Nursing and Midwifery Education and Practice, Enugu

Campus, July 11-12, 2007.

[6] U.I. Nwagwa, Recent trends in the management of

malaria in pregnancy, in: West African College of

Nursing Malaria in pregnancy: Implication for Nursing

and Midwifery Education and Practice, Enugu Campus,

July 11-12, 2007.

[7] Lagos State Ministry of Health Malaria Control

Programme 2011, Lagos State Ministry Health 2011,

http://www.malariacontrolprogram.htm (accessed Dec.

22, 2011).

[8] C.F. Curtis, B. Jana-Kara, C.A. Maxwell, Insecticide

treated nets: Impacts on vector populations and relevance

of initial intensity of transmission and pyrethroid

resistance, J. Vector Borne Dis 40 (2003) 1-8.

[9] WHO, The global strategic plan 2005-2015, WHO

Geneva, 2005, http//www.rollbackmalaria.org/forum

/docs/gsp_en.pdf (accessed April 8, 2011).

[10] World Health Organisation, Roll Back Malaria 2005,

World Malaria Report, WHO Geneva, 2005.

[11] C.I. Tobin-West, B.A. Alex-Hart, Insecticide treated bed

nets ownership and utilization in River State, Nigeria

before a state wide net distribution campaign, J. Vector


1123

Borne. Dis. 48 (2011) 133-137.

[12] A.Y. Isah, E.I. Nwobodo, Awareness and utilization of

insecticide treated nets among pregnant mothers at a

tertiary health institution in north western Nigeria, Niger

J. Med. 18 (2009) 175-178.

[13] U.M. Chukwouocha, I.N.S. Dozie, C.O.E. Onwuliri, C.N.

Ukaga, B.E.B. Nwoke, B.O. Nwankwo, et al.,

Perceptions on the use of insecticide treated nets in parts

of Imo River Basin, Nigeria: Implications for preventing

malaria in pregnancy, Afr. J. Reprod. Health 14 (2010)

117-128.

[14] A. Astatkie, A. Feleke, Utilization of insecticide treated

nets in Arbaminch town and malarious villages of

Arbminch Zuria District, southern Ethiopia, Ethiop, J.

Health Dev 24 (2009) 15-24.

[15] Anti-Malarial Treatment Policy 2005, Federal Ministry of

Health, Nigeria, Government Press, Abuja, Nigeria,

2005.


Spatiotemporal Distribution of Phlebotomine Sand

Flies (Diptera: Psychodidae) in a Focus of Cutaneous

Leishmaniasis in Foum Jamâa (Azilal, Atlas of Morocco)

Hassan Arroub1, Abdelaaziz Alaoui1, Hicham El Miri2, Meryem Lemrani3 and Khalid Habbari1

1. Laboratory of Management and Valorization of Naturals Resources, FST, Sultan Moulay Slimane University, Beni Mellal 23000,

Morocco

2. Faculté des Sciences Agdal, Université Mohamed V Agdal, Rabat 10000, Morocco

3. Laboratory of Leishmaniasis, Institut Pasteur du Maroc, Casablanca 20000, Morocco

Received: March 12, 2012 / Accepted: May 29, 2012 / Published: October 30, 2012.

Abstract: An entomological survey of Phlebotomine sand flies (Diptera: Psychodidae) was carried out in three sectors of Foum Jamâa region (province of Azilal, Morocco) during the year 2010. Morphological identification was performed on a total of 1,152 sand flies (23% females and 77% males) collected by sticky paper traps. 80% of the total collected flies were identified as Phlebotomus (Paraphlebotomus) sergenti (Parrot) (57%) and Phlebotomus (Larroussius) longicuspis (Nitzulescu) (23%). In addition to these dominant species, four other species were found, Phlebotomus (Phlebotomus) papatasi (Scopoli), Sergentomyia (Sergentomyia) minuta (Rondani), Phlebotomus (Larroussius) perniciosus (Newstead) and Phlebotomus (Paraphlebotomus) chabaudi (Croset). Overall, the population dynamics show a yearly bimodal pattern related to rainfall and temperature, and with high density around human dwellings. The spatiotemporal distribution of sand fly species was helpful to discuss strategies that might be useful in controlling cutaneous leishmaniasis transmission in this endemic focus. Key words: Phlebotomine sand flies, cutaneous leishmaniasis, temporal and spatial distribution, Foum Jamâa, Morocco.

1. Introduction

Cutaneous leishmaniasis (CL) is endemic in large

parts of Morocco and is caused by transmission of the

parasite Leishmania through the bite of infected

female Phlebotomine sand flies (Diptera: Psychodidae,

Phlebotominae, Phlebotomus) [1, 2]. From a public

health perspective, this is one of the most important

emerging infectious diseases and was included in the

list of notifiable diseases in Morocco. The total CL

cases reported in 2008 were 5,128 against 655 cases in

1998, showing a clear tendency for an increase in the

last 10 years [3]. In Morocco, CL caused by

Leishmania major has been reported since 1914 [4],

Corresponding author: Abdelaaziz Alaoui, Ph.D., research fields: population genetics, molecular biology. E-mail: [email protected].

whereas, the first case due to Leishmania tropica has

been reported in 1989 [5], more recent studies have

incriminated L. infantum as causative agent of CL [6].

In the review of Kimutai et al. [7], the authors

recommended the need for additional epidemiologic

and ecologic studies of CL in conjunction with species

identification.

In the province of Azilal, Foum Jamâa (FJ) region

has become an epidemic focal point for CL. In our

previous work foxed on the eco-epidemiological and

socioeconomic study, the CL consists of a rural

domestic form, which is characterized as affecting the

whole family nucleus, without distinction in sex, due

to the fact that dwellings are located near the natural

focus of transmission, thus propitiating the vector’s

arrival in the house [8]. Geographically and

Spatiotemporal Distribution of Phlebotomine Sand Flies (Diptera: Psychodidae) in a Focus of Cutaneous Leishmaniasis in Foum Jamâa (Azilal, Atlas of Morocco)

1125

socio-economically, FJ region constitutes a crossroads

of several human daily activities in the province of

Azilal and a bridge between two of the main towns of

Morocco (Beni Mellal and Marrakech). During four

years (2006-2009), the authors registered about 500

cases of CL in this region [8].

In Morocco as in many other countries: Saudi

Arabia, Ethiopia, Turkey and Israel, Phlebotomus

sergenti Parrot has been shown to transmit L. tropica

[2, 9-12]. In addition, Phlebotomus papatasi Scopoli

was proven to be a vector of L. major in Morocco [4].

The distribution of sand flies is due, in great part, to

the bioclimate [13]; the effect of altitude factor was

demonstrated [14]. In many parts of the world, it is

suspected that the reemergence of the CL, in the last

few decades, is associated indirectly to human

activities that cause environmental changes, favorable

to both the reservoir and the vector [15, 16]. In FJ

region, rapidly changing environmental conditions

caused by habitat destruction, such as that associated

with deforestation/urbanization processes could have

an enormous influence on vector populations and

consequently on disease transmission. There was no

previous study on the diversity and the abundance of

sand flies species within this region; while some CL

vector species may disappear, others may become

more abundant, additionally, vector species that

hitherto had no presence in these zones can arrive,

adapting to anthropogenic environmental conditions.

All of the above makes it necessary to evaluate the

sand fly fauna and their distribution, to elucidate

vector behavioral habits, population’s densities,

seasonal dynamics, feeding preferences or insecticide

susceptibility patterns, which are the most important

aspects for disease control. The lack of data on the

sand flies diversity in this endemic region led us to

initiate this study, which aims as a first step, to carry

out a yearly entomological survey to determine: (1)

the taxonomic composition of sand fly species vector;

(2) the spatial and temporal distribution of the CL

vector. The location selected for collections was the

same one in which the first study on CL had been

done. This allowed us to shed more light on the cycle

of the vector and designate factors responsible for the

transmission of such disease as CL, both specific to

the region studied. Results should help us to elaborate

an effective program with more specific preventive

actions such as the period of the year appropriate for

insecticide treatment and the biotope that should be

targeted during treatment.


2.1 Study Sites

The study was conducted in Foum Jamâa region,

the province of Azilal, Atlas of Morocco (Fig. 1). The

area was subdivided into 3 adjacent sectors, namely,

Fig. 1 Map of the study area showing the position of the Foum Jamâa region (province of Azilal) in Moroccan map, the surfaces of the sectors (Beni Hssan, Foum Jamâa) are indicated in bracket below each corresponding sector’s name.


1126

Foum Jamâa (FJ), Beni Hassan1 (BH1) and Beni

Hassan2 (BH2), overall cover about 400 km2. This

region is located near the Western High Atlas National

Park (32°08′ N, 6°60′ W) at different altitude from

523 m to 1,086 m. The agriculture remains the

primary source of income and is mainly based on the

production of wheat, almond, olive and lentils;

domestic animals include rearing of chicken, sheep,

cattle and horses. In addition, most homes have at

least one dog. The villages are surrounded by

mountains with some natural water sources and cactus,

which serve as refuge for rodents and sugar source for

sand flies. Excepting the sector of FJ, in which some

modern houses exist, traditional constructions are the

main type of habitat in the study area.

2.2 Monitoring Sand Flies

Sand flies monitoring was conducted on a total of

17 stations representing 8 main different biotopes in

the region of FJ (henhouse, stable, cave, ruined house,

dwelling, bridge, windows, outdoor) and regularly

distributed to cover all supervised area. A one year

(2010) collection was made biweekly using sticky

paper traps (21 cm × 27.3) impregnated with grapes

oil. The same number of traps (6 traps) was installed

for each station; they were recovered after one night

of survey (between 6 p.m. and 8 a.m.). Collected sand

flies were placed in glass vials containing 70% ethanol.

After sex determination, all collected sand flies were

mounted on glass slides, using Canada balsam, and

were identified by morphological taxonomic keys

according to Boussaa et al. [17]. Both preparation and

identification of specimens were made individually.

Monthly temperatures and rainfall were provided by

the regional department of Moroccan national

meteorology.


Data were subjected to one-way analysis of

variance (ANOVA) in order to determine the

significance of differences of total collected sand

flies between sectors, months and biotopes. Shannon

information index based on the matrix of

presence/absence of each sandfly species was used to

compare species diversity between sectors,

calculated as: H = -Σ1S (piln(pi)). With ni: the number

of individuals in species i or the abundance of

species i; S: the number of species, also called

species richness; N: the total number of all

individuals; pi: the relative abundance of each

species, calculated as the proportion of individuals of

a given species to the total number of individuals in

the community: ni/N. This parameter allowed as to

compare between sectors by a single diversity index

which take into account even species abundance and

species richness.

3. Results

3.1 Sandfly Species Diversity

In this study, a total of 1,152 sand flies were

collected using 2,442 sticky traps from FJ region

throughout the years 2010. Overall, six species were

identified, consisting of five Phlebotomus spp. and

one Sergentomyia (Table 1). The most dominant

species was P. (Paraphlebotomus) sergenti (Parrot)

(57%), followed by P. (Larroussius) longicuspis

(Nitzulescu) (23%). These two abundant species

constituted 80% of the total collected flies. P.

chabaudi was the rarest species represented by one

individual. The sex-ratio indicated that more males

were collected than females for all species. The

overall male/female ratio was 3.3:1. These traps also

Table 1 Sand fly species collected in Foum Jamâa region (Azilal, Atlas of Morocco) and their relative abundance.

Sand fly species Male Femelle Total %

P. papatassi 51 24 75 7

P. sergenti 486 176 662 57

P. longicuspis 229 31 260 23

P. perniciosus 48 1 49 4

S. minuta 71 34 105 9

P. chabaudi 1 0 1 0

Total 886 2 1,152 100


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collected large numbers of non-target organisms,

including non-biting flies and Lepidoptera.

3.2 Spatial Distribution of Sand Flies

Occurrence of sand flies species in the three sectors

of FJ was reported in Fig. 2 and Table 2. One factor

ANOVA indicates a significant difference (P ≤ 0.01)

between the three sectors. BH2 was the most infested

sector (n = 677, 59%), followed by BH1 (n = 365,

32%) and FJ (n = 110, 10%) was the lowest colonized.

This was valid for all sand flies species except S.

minuta which was more abundant in BH1 than in BH2.

More accurate comparison of sectors in terms of

number of sand flies collected must take into account

the number of traps recuperated from each sector. The

mean number of sticky traps was not significant

between the three sectors (P = 0.06; Table 2). To

catch one sandfly, in the same above experimental

conditions, we have to put respectively 10, 2 and 1

traps within each of the sectors: FJ, BH1 and BH2. In

Fig. 2 Sand fly species collected in the three sectors of Foum Jamâa region, Azilal, Atlas of Morocco. FJ: Foum Jamâa; BH1: Beni Hassan sector 1; BH2: Beni Hassan sector 2.

general, no dependence between species and sector,

except for P. chabaudi that was represented by one

individual in the sector BH1. All sand flies species

were present in the three sectors, Shannon information

index, which varied from 0.16 to 0.41, indicates that

the BH2 sector was the most diverse (Table 2).

3.3 Temporal Distribution of Sand Flies

Monthly monitoring of sand flies vector activity

during the 1-year-study is shown in Fig. 3. A total of

1,152 sand flies were collected from January to

December 2010. ANOVA has showed that the number

of recovered traps between the twelve months was not

significant (P = 0.08), and the temporal fluctuation of

population density was due to the seasonality. Sand

flies flight activity of total population was strongly

marked during two periods: the most important one,

started in the end of March and reached a peak in June

for all sand flies species except S. minuta which

peaked during July, while the activities of the other

Fig. 3 Monthly temporal patterns of sand flies species in Foum Jamâa region (province of Azilal, Atlas of Morocco) during the year 2010.

Table 2 Spatial distribution and diversity of sand flies collected from three sector of Foum Jamâa region (Azilal, Atlas of Morocco).

Sector Traps recovered Collected sand flies

NS/NT Shannon index

NT % Mean* NS % Mean** Mean SE

FJ 1,129 46 5.48a 110 10 0.51a 0.10 0.16 0.06

BH1 642 26 5.58a 365 32 3.15b 0.57 0.33 0.07

BH2 671 27 5.89a 677 59 5.94c 1.01 0.41 0.10

Total 2,442 100 5.61 1,152 100 2.63 0.47 0.30 0.05

NT: number of traps; NS: number of sand flies; for each column, different letters a, b and c indicate that the means are significantly different.* P = 0.06; ** P ≤ 0.01. FJ: Foum Jamâa; BH1: Beni Hassan sector 1; BH2: Beni Hassan sector 2.


1128

species decreased to a minimum during this month.

The second period of intense sand flies activity

corresponds to August-October-November depending

on sand flies species and falls to low levels in the end

of November. During the months January to March

and December no sand flies were captured. To

understand the conditions that determine this temporal

distribution of sand flies, we collected data of rainfall

and temperature in the region of FJ, during 2010.

Fig. 4 shows the monthly cumulative rainfall (mm)

and the mean monthly temperatures in this region. The

maximum and minimum mean monthly temperatures

were respectively 45 °C (July) and 10 °C (December),

and the rainfall was relatively low with two peaks: one

in autumn (60 mm) (October to December) and the

other in spring (100 mm) (March and April).

3.4 Sand Flies Occurrence According to Biotope

The distribution of the collected total number of

sand flies according to biotope was shown in Fig. 5.

The highest number of sand flies captured was

observed in the stable with a value of 648 (56%)

followed by dwelling, cave and stable-henhouse with

about 14-10%, then windows, bridge, ruined house,

henhouse and outdoor with less than 25 (2%) sand

flies for each one. These results were valid for all sand

flies species except for S. minuta which prefers the

cave than stable and dwelling. Before interpreting

these results, one should take into account the result of

the one-way ANOVA applied for variable number of

Fig. 4 Mean monthly temperature T (°C) and rainfall P (mm) during 2010 around the study area of Foum Jamâa, province of Azilal, Atlas of Morocco.

traps and number of sand flies. The number of

recovered traps according to biotope factor was highly

significant (P ≤ 10-4) and the mean number of sand

flies collected was also highly significant (P = 0.0013).

The effect of biotope, although it interferes with the

number of recovered sticky traps, was a significant

factor that influences spatial distribution of sand flies.

The stable biotope even had the significantly greater

number of recovered traps however did not have the

greater number of captured sand flies. Fig. 6 shows

the number of sand flies collected per biotope and

explains more clearly that the dwelling was the first

preferred biotope with population sand flies density

that might reach two folds of the other biotope

(Student’s t test, P = 0.0013). The second group of

Fig. 5 Sand flies species distribution according to different biotope of Foum Jamâa region (province of Azilal, Atlas of Morocco) during the year 2010.

Fig. 6 Number of collected sand flies per sticky trap installed in different biotopes during 2010 in the region of Foum Jamâa, province of Azilal, Atlas of Morocco.


1129

favorite biotopes were stable, cave and stable-henhouse,

and significantly a lower number of captured sand

flies were registered in the other biotopes (windows,

bridge, ruined house, henhouse and outdoor).

4. Discussion

4.1 Sandfly Species Diversity in FJ Region

This study is the first one to deal with the

taxonomic composition and the spatiotemporal

distribution of sand flies at the micro-geographical

level in FJ region. The distribution and incidence of

leishmaniasis are both influenced by human behavior

and environmental variables affecting the vector and

the reservoir populations. In FJ, the poor surveillance

of the vector complicates the planning and

implementation of control strategies.

During a single year, twice a month, in an altitude

ranging between 532 m and 1,086 m, the study of the

sandfly fauna in FJ region identified six species. All

of them are included in the list of 22 phlebotomine

species existing in Morocco [18]. In order of

abundance, P. sergenti comes the first with 57%, this

is usually a dominant sandfly species in foci of

anthroponotic CL in the Old Word [19, 20] and the

study area is known to contain the highest genetic

diversity of this species [21]. Phlebotomus sergenti is

a main vector of L. tropica in Morocco, although in

some specific areas (Saudi Arabia; Palestinian

Territories) other sand flies can play a role in L.

tropica transmission [9, 12]. The second more

abundant species was P. longicuspis representing 23%

of the total capture. Among the six species, P.

papatasi is known to be widely distributed throughout

the world and often abundant [22] and was proven to

be a vector of CL due to L. major [19]. Phlebotomus

chabaudi was also suspected as vector of L. tropica

[23].

Several types of traps are described in Refs. [24,

25], and most traps are adapted to very specific

questions. Sticky papers are one of the standard

surveillance techniques that have been used to collect

adult phlebotomin sand flies. Collectors are less

exposed to the risk of leishmania infection. However,

these traps often yield small or no catches [26], so FJ

region could include a greater density than that

observed in our study. Maroli et al. used sticky traps,

hand aspiration and CDC traps, and they choose to

analyze sticky paper data to determine the seasonal

trends in the sandfly density [27].

Sand flies collection with sticky traps caught more

males than females, this confirms data obtained

elsewhere using the same type of traps [28], also

Feliciangeli et al. [29] found that males were almost

twice abundant as females, while using CDC light

traps, it is found that males sand flies account for 79%

of the catch [30]. Whereas, there were no significant

differences between the number of males and females

caught by light traps [31], several other studies used

traps (different combinations of visual and chemical

attractive features) that captured significantly more

females than males [32, 33]. Our results could be

explained by the type of traps used rather than the

disproportion in the natural sex-ratio of sand flies

captured in FJ region. In addition, since the dispersion

capacity is reduced for males than for females [34]

due to the fact that females need blood meals for egg

production every few days [35], and sand flies are

generally considered to be poor fliers, not traveling

from their breeding and resting sites [19], therefore,

the high number of captured males provides evidence

of the presence of sand flies breeding sites around

human habitations in FJ region, and that

females-specific traps would be desirable for the

control of sand flies around houses.

FJ region shows high biodiversity of phlebotomine

fauna, with six species among 22 identified in

Morocco [18]. Shannon information index takes into

account the number of species and their frequency, so

this index expresses more correctly the diversity of

each sector. It was concluded clearly that the BH2

sector was the most diverse. The general theories

stipulated that diversity usually is reduced gradually


1130

form population origin to newly colonized areas. It

appears that BH2 contains the reservoir of the

population source. Preventive actions undertaken by

the national program of leishmaniasis control,

supervised by the Moroccan Ministry of Health,

should be more focused in these zones.

4.2 Temporal Distribution of Sand Flies

Sand fly seasonal trends in density expressed as the

number of sand flies collected have showed two peaks,

one in June and another late in August. This bimodal

profile was nearly the same as those observed in other

previous studies in different parts of the world; in

Syria for P. sergenti and P. papatasi [27] and in

Morocco for P. papatasi, S. minuta and S. fallax [36].

During the months January to March and December,

no sand flies were captured, due to severe weather

conditions, which occurred in the region of FJ. The

summer of 2010 in Moroccan was very hot, maximal

temperature recorded in July reached 50°C and above;

this explains the fall of population density of sand

flies. When extreme temperatures were registered

during the survey days or the month prior to sampling,

vector activity and thus vector captures were limited

[37]. The temperature is one of the main factors

preventing the spread of leishmaniasis to northern

Europ [38]. Quate [39] classified the sand flies of the

southern Sudan into two groups: “seasonal species”

that appear only during the dry season, and

“non-seasonal species” that exist throughout the

whole year. Unlike the profile of seasonal occurrence

of sand flies obtained in other parts of Morocco, in the

urban area of Marrakech, for example, P. papatasi is

active throughout the year [36]; no one of the six

species identified in FJ region was active throughout

the year 2010.

4.3 Sand Flies Occurrence According to Biotope

Sand flies occurrence according to biotope shows

that the dwelling was the favorite biotope at least for P.

sergenti and P. longicuspis, the limited number of

catch does not allow to clarify the preferred biotope of

the other species. Phlebotomus papatasi has an

endophilic distribution; it is largely found in domestic

and peri-domestic habitats [40] and is an aggressive

man biter [22]. In arid environments, this species is

rarely found far from rodent burrows or human

habitations [41]. P. longicuspis is also an

anthropophilic species that prefers biotopes

characterized by dense vegetation, close to human

dwellings [42].

5. Conclusions

Based on the monthly collections of sand flies over

one year in the FJ region where CL is known to be

endemic, we suspect the incrimination of at least P.

sergenti and P. longicuspis in the CL transmission in

FJ. In addition to the endophilic character, the

presence of attractive vegetation, such as cactus [33],

and the rural lifestyle enhance the presence of the

resting and the breeding sites of the vector sand flies

around the human dwelling.

Since most control efforts against sand flies are

aimed at interrupting contact between female sand

flies and human, several methods were used to

accomplish this goal. Sand flies generally are highly

susceptible to insecticides but it depends on the

manner of exposure and contact [43]. Residual

insecticide house-spraying has been used successfully

against endophilic species [44]. Insecticide-

impregnated curtains, bed nets, or bed covers were

also used but with limited success against sand flies

[45]. Barriers may be useful to stop incoming sand

flies; In French Guiana, clearing the forest around a

village, and fogging with insecticide to a radius of 400 m

reduced the sand fly density and the incidence of

leishmaniasis significantly, but it is not an acceptable

method since it causes considerable damage to the

environment [46]. In the region of study, further

knowledge on resting and breeding sites of sand flies

are required to elaborate a plan to attack the adults or

immature stages at outdoor sites. Also further


1131

investigations on isolating leishmania strains from

vectors are needed to clarify the CL transmission risk,

and to confirm the role of P. sergenti and probably the

involving of P. longicuspis in the transmission of

Leishmania in this CL focus. These will be the

prospect aim in our group.

Acknowledgments

The authors are very grateful to the communities

and the health district authorities of Foum Jamâa for

their support of this study. The authors also thank the

English teacher Mr. Rahhal Ajbilou (Oued Alabid

High School) for linguistic correction.

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[34] B. Yuval, A. Warburg, Y. Schlein, Leishmaniasis in the Jordan Valley, V. Dispersal characteristics of the sandfly Phlebotomus papatasi, Med. Vet. Entomol. 2 (1988) 391-395.

[35] R. Killick-Kendrick, The biology and control of phlebotomine sand flies, Clin. Dermatol. 17 (1999) 279-289.

[36] S. Boussaa, S. Guernaoui, B. Pesson, A. Boumezzough, Seasonal fluctuations of phlebotomine sand fly populations (Diptera: Psychodidae) in the urban area of Marrakech, Morocco, Acta Tropica 95 (2005) 86-91.

[37] R. Galvez, M.A. Descalzo, G. Miro, M.I. Jimenez, O. Martin, F. Dos Santos-Brandao, et al., Seasonal trends and spatial relations between environmental/meteorological factors and leishmaniosis sand fly vector abundances in central Spain, Acta Trop. 115 (2010) 95-112.

[38] K.G. Kuhn, Global warming and leishmaniasis in Italy, Bull. Trop. Med. Int. Health 7 (1999) 1-2.

[39] L.W. Quate, Phlebotomus sandflies of the Paloich Area in the Sudan (Diptera, Psychodidae), J. Med. Entomol. 1 (1964) 213-268.

[40] L. Orshan, D. Szekely, Z. Khalfa, S. Bitton, Distribution and seasonality of Phlebotomus sand flies in cutaneous leishmaniasis foci, Judean Desert, Israel, J. Med. Entomol. 47 (2010) 319-328.

[41] G. Wasserberg, Z. Abramsky, G. Anders, M. El-Fari, G. Schoenian, L. Schnur, et al., The ecology of cutaneous leishmaniasis in Nizzana, Israel: Infection patterns in the reservoir host, and epidemiological implications, International J. Parasitol. 32 (2002) 133-143.

[42] S. Guernaoui, A. Boumezzough, Habitat preferences of phlebotomine sand flies (Diptera: Psychodidae) in southwestern Morocco, J. Med. Entomol. 46 (2009) 1187-1194.

[43] L. Orshan, D. Szekely, H. Schnur, A. Wilamowski, Y. Galer, S. Bitton, et al., Attempts to control sandflies by insecticide-sprayed strips along the periphery of a village, J. Vector Ecol. 31 (2006) 113-117.

[44] J.B. Vieira, G.E. Coelho, Visceral leishmaniasis or kala-azar: The epidemiological and control aspects, Rev. Soc. Bras. Med. Trop. 31 (1998) 85-92.

[45] O. Courtenay, K. Gillingwater, P.A. Gomes, L.M. Garcez, C.R. Davies, Deltamethrin-impregnated bednets reduce human landing rates of sandfly vector Lutzomyia longipalpis in Amazon households, Med. Vet. Entomol. 21 (2007) 168-176.

[46] B. Alexander, M. Maroli, Control of phlebotomine sand flies, Med. Vet. Entomol. 17 (2003) 1-18.


Determination of Fungal Colonization in the Oral Cavity

of College Students

Floridia Ricardo, Rodriguez Graciela, Ampuero Verónica and Gonzalez Luis

Department of Parasitology and Mycology, National University of San Luis, San Luis 5700, Argentina


Abstract: In recent years, an increase in opportunistic fungal colonization in the oral cavity in immunocompetent patients (IC) has been observed. In the bibliography, the most observable genre is Candida and less frequently found are other opportunistic such as Aspergillus, Rhizopus, Cryptococcus, and others. The authors determined the presence of fungi in the oral cavity of IC students, and their relationship with the concentration of secretory IgA in saliva. To this end, we collected 50 samples of oral cavity swabs, which underwent direct examination and culture in Sabouraud dextrose agar with chloramphenicol. For its identification, CHROMagar Candida and API Candida (BioMerieux) were used. We obtained nine positive cultures (7 Candida albicans and 2 Saccharomyses cerevisiae), which represented 18% of the studied population. Throughout radial immunodiffusion (RID plates-PLATE), we determined the concentration of secretory IgA. No relationship was observed between the colonized group and group that was not colinized. The colonization rate found is below the one described in the bibliography (30% to 50%). However, these opportunistic fungi cause transitory colonization with no clinical relevance in IC patients and, its percentage can vary according to the studied population. Key words: Oral cavity, immunocompetent, Candida, fungal colonization.

1. Introduction

In recent years, there has been an increase in the

development of fungal infections in the oral cavity of

immuno-compromised patients [1, 2]. Among the

causative organisms, we can include Aspergillus,

Mucormycosis, Rhizopus, Cryptococcus and other (all

observed at low frequencies), but undoubtedly the

most common genus is Candida. In

immuno-competent people but not an increase in the

number of frames fungal disease, an increase in the

colonization of these opportunistic fungi has been

observed.

Candidiasis is a cosmopolitan disease of great

importance and one of the most frequent fungal

infections in the oral cavity. It affects both sexes

equally and has no preference of age, except the one

Corresponding author: Floridia Ricardo, biochemist,

laboratory assistant, research fields: parasitology and mycology. E-mail: [email protected].

observed at the ends of life. At present, its incidence is

increasing.

Thrush is characterized by the appearance of a

creamy white stippling or yellowing of the mouth

(gums, tongue, oral mucosa or corners), which is

slightly over-large, and can cause pain. When the

condition is more intense, instead of dotted there are

large white patches on the tongue and the rest of the

mucosa of the mouth, the rub is eliminated or plates

and stippling are superficial wounds that bleed slightly

[3-5].

Organisms causing this disease belong to the genus

Candida, which consists of yeasts ovoid 2-6 m wide

× 3-9 m long, are Gram positive, and that after 48 h

of culture in Sabouraud media as added or no

antibiotic (chloramphenicol or gentamycin), colonies

of 4 to 5 mm in diameter, smooth, glossy or matt

white color. Cultivation alone will inform us of the

existence of yeast, but not differentiate colonization

Determination of Fungal Colonization in the Oral Cavity of College Students

1134

from infection, so the observation of a direct

examination is essential. If the same individualize a

pseudomycelia (C. glabrata does not pseudomycelia)

and inflammatory cells we will suggest an infection, if

blastospores are seen going to suggest a settlement.

The culture allows the isolation of the fungus for

antifungal susceptibility testing, and thereby applies a

proper therapy on the patient to prevent the selection

of resistant strains. The genus Candida is comprised of

more than 200 species, but this condition only reverts

importance as follows: C. albicans, C. tropicalis, C.

krusei, C. glabrata, C. guillermondi and C.

parapsilosis. The most important is C. albicans that is

isolated by more than 80% of cases; C. tropicalis and

C. glabrata by 10% and the rest are rarely isolated.

Species of the genus Candida are pathogenic in

nature, as diners are part of the ancillary or

supplementary oral microbiota and imbalances need to

occur both in the patient’s immune system and in the

protective flora present in the tissue, to colonize,

infect and cause disease. Within the oral cavity of

Candida species are the most location on the tongue

but they can also live in gums, mucosal wall of the

mouth and palate. They have a great capacity for

adhesion to both host cells, as inert materials

(prosthesis).

Estimated 30% to 50% of healthy people have this

fungus as part of their normal oral microbiota [6-9].

The transformation from commensal to pathogen

depends on the interaction and modification of three

groups of factors. The first factor is the one that

involves the host, in which alterations of the mucosal

barrier, either by maceration, trauma, oral hyperplasia,

epithelial dysplasia and occlusion predispose to

candidiasis, or immune disease (HIV), neoplasms,

deficiency anemia, stress, depression and

physiological changes (pregnancy). The second factor

is the one that involves changes in the microenvironment

of the oral cavity, whose protective commensal flora is

affected by the use of drugs (antibiotics,

immunosuppressants, antineoplastics, psychotropic

drugs, hormones) that sweep the protective

microorganisms away and allow the opportunistic

pathogens to proliferate. The third group of factors is

dependent Candida, given its ability to adhere to the

cells of the mouth or dentures, to evade host defenses,

secrete enzymes such as proteases, phospholipases

and the ability to compete with other microorganisms

for nutrients [9, 10].

The transformation from commensal to pathogen

takes to convert morphogenetic organism in the yeast

phase of the filament. At this late stage, it activates the

pathogenic mechanisms of this fungus that causes the

breakdown of balance between health and

disease [11, 12].

Other elements that allow the host defense of the

proliferation of pathogens in the oral cavity are the

patient’s saliva. It constitutes a primary antifungal

because it has the function of mechanical sweeping

and protein components such as lysozyme, lactoferrin,

lactoperoxidase, glycoproteins and a pH between 5.6

and 7.8 that hinder the adhesion and growth of the

fungus. Anti-yeast are also immersed in the same oral

cavity, they are mostly of secretory IgA and act by

recognizing epitopes on the surface of the fungus,

thereby inhibiting the adhesion thereof to the oral

mucosa, and enabling phagocytosis by PMNs that

possess receptors Fc portion of these antibodies [13, 14].

It is described in the bibliography that patients with

oral candidiasis show increased levels of secretory

IgA in saliva, but Candida can produce proteases that

destroy the antibodies. The determination of IgA in

saliva will inform us whether the immunological

capacity of the patient’s oral mucosa is normal or

insufficient [15, 16].


2.1 Study Area

Fifty samples were collected for mycological study

in oral cavity, from students in the course of

Parasitology and Mycology, Faculty of Chemistry,


1135

Biochemistry and Pharmacy, Universidad Nacional de

San Luis, with an average age of 24 in the participants.

Before developing the work, a briefing was made on it,

and the ones who agreed to participate expressed their

willingness to do so by informed consent, which were

given full details of the work (Ordinance No. 005/08

UNSL).

2.2 Use of the Survey

Participants were given a survey to assess issues

related to conditions affecting the immune system,

either as the taking of medication (corticosteroids,

antibiotics, oral contraceptives, etc.), underlying

diseases (diabetes, anemia, etc.) and alterations that

had previously been suffered in the mouth (thrush,

herpes, gingivitis, bleeding, and others). This survey

also assessed oral hygiene, which was classified into

four groups: performed after each meal, twice daily,

once daily and unrealized. It was inquired if the

participant had the smoking habit, considering

smoking people to those who consumed at least one

cigarette in the month that preceded the study, and

finally if the mentioned participant was completing a

pregnancy period. This survey detailed the current oral

cavity lesions observed during sampling (Appendix A).

2.3 Collection of Samples

The sample collection was conducted in the

Laboratory of Parasitology of the University. At first,

the participant was offered a clean container to collect

the saliva sample for assay of secretory IgA.

Subsequently, he was given water to rinse the mouth,

and to make appropriate swabs. Cotton swabs were

used, with which toured areas of the oral cavity are

more likely to be colonized by yeast according to the

bibliography consulted. Samples were taken from the

surface of gums, palate, tongue and inner walls of the

oral cavity. The swab was immediately sown in Petri

dishes in an agar prepared for this purpose. The

culture agar used was Sabouraud dextrose agar

(Britain) that, once prepared and sterilized, was added

5 mL of Chloramphenicol Succinate (Quemicetina of

Rontag) to achieve a final concentration of 250 mg/L

of the antibiotic in the finished agar. Subsequently, a

second swab from the same areas was conducted and,

with this swab, a mark was prepared on a microscope

slide and then it was coloured by Giemsa staining [17].

2.4 Methods Used

The Petri dishes were cultured in an oven at 28 °C

for 15 days, those in which no growth was observed

in that period of time were reported as negative; on

the contrary, those presenting colonies were tested to

be typed. First, there was a Giemsa stain of

microorganisms that formed the colony, and then

rang in a differential medium (Candida, Paris France)

that allowed the offense; those who could not be

identified are evaluated by Api Candida

(BioMerieux).

In the saliva sample, we came to the

determination of the concentration of secretory IgA

by radial immunodiffusion according to Mancini

technique. Procedure was used for RID

plates-PLATE by placing 5 L of the saliva sample

into each well. The diffusion time was 72 h, after

the stipulated time was over; we measured the halo

of precipitation which correlated with the

concentration of antibody.

All the data was entered in an Excel 2000

spreadsheet program and processed with it.

3. Results

3.1 Obtained Positive Results

Of the 50 samples that were evaluated, from

immuno-competent university students, the growth of

at least one yeast colony was observed in nine of them

(Fig. 1). After being typified finds that seven were

yeasts of Candida albicans species and the remaining

two Saccharomyces cerevisiae (Figs. 3-5).

Regarding the survey data, the following results

were obtained.


1136

Positive 9

18%

C. albicans 7

78%

Negative41

82%

Saccharomices cerevisae

2

Fig. 1 Positivity of the samples studied.

Masculinos 3

33%

Female 6

67%

Fig. 2 Positive cultures according to sex.

41

05

1

0

7

1

03 0 0

31

52

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

Corticoteroids Antibióticts Anticonceptives Levothiroxine Withous

Medication

All Participants

C. albicans

S. cerevisiae

Fig. 3 Participants according to crops exposed to medication.


1137

Smoking 1

11%

No Smoking 8

89%

Fig. 4 Crops according to habits of smoking.

1

0

0

5

1

0

3

0

0

1

0

0

40

6

2

0 5 10 15 20 25 30 35 40

Diabetes

Anemia

Hormonal

Celiac

Disease ‐ free

S. cerevisiae

C. albicans

All Participants

Fig. 5 Positive cultures according to underlying disease.

3.2 Positive Cultures According to Sex (Fig. 2)

3.3 Participants According to Crops Exposed to

Medication (Fig. 3)

The results of fungal colonization obtained in

participants who were exposed to medication were as

follows:

Subject to steroids: 25% (colonized by Candida

albicans);

Subject to antibiotics: 20% (colonized by

Candida albicans);

Subject to contraception: 14% (colonized by

Candida albicans);

Subject to levothyroxine: (0% colonized);

Participants without medication: 16% (colonized

by Candida albicans);


1138

6.45% (colonized by Saccharomyses cerevisiae).

3.4 Crops According to Habits of Smoking (Fig. 4)

The colonization that was obtained in relation to

diseases that the participants could present was:

Disease-free basis: 15% colonization against

Candida albicans and 5% against colonization

Saccharomyses cerevisiae;

Celiac disease: 0% colonization;

Hormonal: 0% colonization;

Anemia: 20% against Candida albicans

colonization;

Diabetes: 0% colonization.

3.5 Positive Cultures According to Underlying

Disease

The colonization percentages that were obtained

when the authors evaluated the oral hygiene are shown

in Fig. 5.

3.6 Positive Cultures According to Oral Hygiene (Fig. 6)

In those participants where oral hygiene was more

frequent (after every meal), the percentage of

colonization that was obtained was 24% to Candida

albicans. While those who conducted the oral cleaning

twice a day, 9.5% was obtained compared to

Saccharomyses cerevisiae. When it was realized once

a day, it showed a 25% colonization of Candida

albicans. There were no participants who did not

conduct oral cleaning at least once daily.

3.7 Relationship between Crops and the Values of

Secretory IgA (Fig. 7)

In relation to the levels of secretory IgA that were

obtained, compared to fungal colonization that was

observed in the oral cavity, the results were as

follows:

In 49 participants the results of secretory IgA that

were found were within the reference values for the

population studied (3 to 20 mg/dL). The average

value in non-colonized participants was of 7.29

mg/dL with a deviation of ± 3.52 mg/dL, while the

population that had some fungal colonization was

11.0 mg/dL ± 6.6 mg/dL.

3.8 Relationship between Sensitivity of Cultivation

and Direct Observation (Fig. 8)

In assessing the sensitivity of culture versus direct

observation (smear) after Giemsa staining (Fig. 2),

the following results were yielded:

25

6

0

21

02

4

10000

0

5

10

15

20

25

D/ Food2 Time per day1 time per dayNot done

Total de participantes

C. albicans

S. cerevisiae

Number o

f Particip

ants

Fig. 6 Positive cultures according to oral hygiene.


1139

Fig. 7 Relationship between crops and the values of secretory IgA.

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

42

44

46

1 2 3 4 5 6 7 8 9

Number of positives cultures

Colonies obtained in

cultures

‐1

0

1

Cultivo Directo

Positive

Positive d

irect

examinatio

n

Positive d

irect

examinatio

n

Negative

Fig. 8 Relationship between sensitivity of cultivation and direct observation.

Of the nine positive cultures, only 7 direct presence

of yeast was observed (77.7% positive). In no direct

the presence of pseudo-mycelium was observed.

4. Discussion and Conclusions

The percentage of fungal colonization that was

obtained in this study was 18%, far below the one

reported in the bibliography (30% to 50%). Although

the population studied is composed of individuals

immunocompetent today, they suffered a fungal

colonization at some stage in their lives, which were

more vulnerable to them. This colonization occurred;

the fungus became part of the microbiota of the oral

cavity, maintaining a constant balance between the


1140

factors of the microorganism which tried to develop

the disease and host factors that tried to counter it.

This could account for fungal development in these

patients [10]. In relation to the types of the isolated yeast, we

conclude that the results were adjusted to the

bibliographical statistics, although the predominant

colonizer was Candida albicans, yielded two crops

Saccharomyses cerevisiae, also described in the

bibliography. All the types of isolation obtained were

not important from the clinical standpoint in

immunocompetent patients.

Because the number of samples for survey data was

not significant, we intend to continue this work in

order to obtain conclusive results.

The bibliography shows that the values of

secretory IgA increase against fungal disease, in our

work, (immuno-competent students), secretory IgA

values obtained were normal and there were no

significant differences between the participants who

were colonized and those who were not.

Establishing the diagnosis of fungal colonization is

to highlight the greater sensitivity of culture versus

direct observation after Giemsa, but when evaluating a

patient, the combination of the two techniques

provides us with essential information [16-18].

Acknowledgments

This work was supported by the Universidad

Nacional de San Luis, Faculty of Chemistry,

Biochemistry and Pharmacy, Clinical Analysis Area,

Course of Parasitology and Mycology.

The authors also thank the active participation of

students in the course of Parasitology and Mycology

which contributed greatly to the development of the

work.

References

[1] J.R. Regueiro, C. Lopez Larrea, Immunology, in: Biology and Pathology of the Immune System, 2nd ed., Pan American Medical Publishing, Madrid, 1997.

[2] I. Roitt, J. Brostoff, D. Male, Immunology, 5th ed., Mosby, London, 1998.

[3] J.L. Puerto, P. Garcia Martos, A. Marquez, L. Garcia Agudo, J. Mira, Oropharyngeal candidiasis, J. Diagn. Biol. Rev. 50 (4) (2001) 177-181.

[4] C. Cortés, Esophageal candidiasis in immunocompetent patients: Clinical and immune systems, Internal Medicine, Hospital Clinico Universidad de Chile, Chile, Medical Journal of Chile 132 (11) (2004) 1-8.

[5] L.J. Lazarde, Erythematous candidiasis of the oral cavity: Case report and literature review, Venezuelan Dental Act 41 (3) (2003) 2-8.

[6] M.M. Panizo, V. Reviákina, Candida albicans and its pathogenic effect on mucous membranes, Come Rev. Soc. Microbiol. 21 (2) (2001) 2-4.

[7] J.L. Port, P. Garcia-Martos, A. Marquez, L. Garcia-Sharp, J. Look, Oropharyngeal candidiasis, Journal of Biological Diagnosis, Rev Diagn Biol 50 (4) (2001) 1-6.

[8] E.I. Cadozo, Host defense mechanisms in sub-prosthetic stomatitis induced by candida, Venezuelan Dental Act 40 (3) (2002) 2-6.

[9] J. Rodriguez Ortega, Candidiasis of the oral mucosa: Literature review, Journal of Dentistry Estomatol 39 (2) (2002) 4-23.

[10] J.M. Aguirre Urizar, Oral candidiasis, Micol Iberoam Rev 19 (2002) 17-21.

[11] G. Quindós, R. Vargas. M. Ruesga Alonso, Processing the samples of the oral cavity and otolaryngology, in: Revista Iberoamericana de Mycology, Chapter 8, 2001.

[12] E. Angoulvant, Rules of interpretation of Candida infections, Bioquím Act. Clinker. Latinoam. 41 (4) (2007) 2-10.

[13] R. Gordillo, Prevalence of Candida albicans isolated from the oral cavity of patients with cancer, Latin American Dental Journal (2) (2008) 38-41.

[14] J.M. Aguirre Urizar, Oral candidiasis, Rev. Iberoam Micol. 19 (2002) 17-21.

[15] C.G. Malbrán, Presumptive identification of yeasts course of medical interest, Argentina Association of Microbiology, ANLIS, Mycology Department, National Institute of Infectious Diseases, 2010.

[16] C. Fernandez, Analysts, Modesto Lafuente, Laboratoire de Parasitologie, Hôpital Tenon, AP-HP, Paris, Published by the Spanish Association of Pharmaceutical Analysts (AEFA) with permission from the French magazine Biologiste et Practicien, Spanish Association of Pharmacists, 3 - 28010 Madrid, Espina, 2007.

[17] W. Quindós, R.A. Vargas, M.T.A. Ruesga, Processing the samples of the oral cavity and otolaryngology, Iberoamenricana Journal of Mycology, Chapter 8, 2007, pp. 1-8.

[18] C.M.U. Churches, Prevalence of Candida species in oral cavity in patients with type 2 diabetes, Ph.D. Thesis, Department of Stomatology, Faculty of Dentistry, University of Granada, Granada, 2008.


1141

Appendix A

Poll

Code:

History of diseases or abnormalities in the mouth:

Taking medication

-Steroids

-Immunosuppressants

-Antibiotics

-Other

Presence of underlying disease

-Diabetes

-Neoplasms

-Immunosuppressive diseases

- Severe hepatic or renal

-Deficiency anemia

-Hormonal

Other conditions

-Pregnancy

-Smoking

-Oral hygiene:

(After c/meal) (2 times daily) (1 time per day) (not done)

- Observation of current injuries


Preliminary Results of Crayfish Distribution and

Diseases in Latvia

Inese Briede

Daugavpils University, Vienibas 13, LV 5400, Daugavpils, Latvia

Received: April 16, 2012 / Accepted: July 12, 2012 / Published: October 30, 2012.

Abstract: A lot of water reservoirs offer good natural conditions for crayfish breeding. Today there are four crayfish species present in Latvia—the European species noble crayfish (Astacus astacus), narrow-clawed crayfish (Astacus leptodactylus), the North American signal crayfish (Pacifastacus leniusculus) and spiny-cheek crayfish (Orconectes limosus). In general, crayfish were found in 258 localities—lakes (175), rivers and streams (66), reservoirs, ponds and gravel-pits (17). A. astacus in Latvia is the dominant crayfish species distributed in all regions. Narrow-clawed crayfish was registered since 1960s. North American signal crayfish was introduced due to its resistance to diseases, but spiny-cheek crayfish arrived from Lithuania waters spontaneously. The main threat for crayfish population is crayfish plague, initiated by mucus Aphanomyces astaci. Astacus astacus was more susceptible species in comparison with Pacifastacus leniusculus and Orconectes limosus. Though the North American signal crayfish and spiny-cheek crayfish were not only resistant, they were the carriers of this disease. The physical habitat alterations, pollution and overfishing are significant during the first year breeding period. Crayfish might serve as bacteria carriers and can cause substantial fish diseases (such as aeromonosis, flavobacteriosis). Key words: Crayfish, crayfish plague, bacteria, population.

1. Introduction

Crayfish farming starts in Latvia at the beginning of

last century due to good natural water and breeding

conditions. There were not systematic data about

crayfish investigations at first but in 1930s and 1960s

the situation changed dramatically because of the

disease, supposedly crayfish plague. This disease

eliminated the largest of native crayfish populations.

The yield has further decreased during the last decades

and there was no legal crayfish catch in Latvia in the

1990s [1, 2].

Today there are recognized four crayfish species in

Latvia—noble crayfish (Astacus astacus), narrow-

clawed crayfish (Astacus leptodactylus), the North

American signal crayfish (Pacifastacus leniusculus)

and spiny-cheek crayfish (Orconectes limosus).

Corresponding author: Inese Briede, M.biol., research

fields: fish and crayfish diseases. E-mail: [email protected].

The noble crayfish is only native species and is

breeding in farms for restocking purposes to renew the

balance of crayfish populations in natural waters.

Regularly from years 1926 till 2005, artificial crayfish

restocking was prepared in 74 lakes. In 2009 the noble

crayfish were found in 129 lakes or 84.9% from all

lakes.

The North American signal crayfish was introduced

in Latvia in 1983 and is breeding in farms for human

consumption due to faster growth and its resistance to

diseases.

Narrow-clawed crayfish was reported since 1960

and this population is distributed in separate water

bodies. In 2009 the narrow crayfish was found in 25

lakes or 16.4% from all lakes.

Spiny-cheek crayfish was found in natural waters in

one locality and it was suggested that it came from

Lithuania waters spontaneously. In 2009 spiny-cheek

population was found in one locality.

Preliminary Results of Crayfish Distribution and Diseases in Latvia

1143

From previous studies it is known that North

American crayfish species are the carriers of the fungi

Aphanomyces astaci the agent of crayfish plague [3],

but the carrier of the disease has not been confirmed in

Latvia.

The objective of this study is to detect freshwater

crayfish populations in natural waters and set out the

future investigations to detect the presence of the

parasite A. astaci in North American crayfish species

that can cause mass mortalities of European native

crayfish in Latvia.


These data have been collected and categorized

from the inquiries and field investigations. In total

there was information on crayfish from 258 localities

in Latvia. Most of them are lakes (175), but crayfish

was registered in rivers and streams (66), in water

bodies, ponds and gravel-pits (17) [4].

Crayfish were collected with traps from June to

October during 2002-2004. Male and female were

taken separately. Weight was measured by placing live

crayfish on absorbant paper for several minutes and

then weighting them to the nearest 0.1 g. Length

measurements include total length—distance from tip

of rostrum to tip of telson with the crayfish placed on

its back. The identification was estimated by

morphological signs. Population was classified as

weak, medium and good according to catch per unit

effort (number of crayfish per trap night: < 0.5 weak,

0.5-2.5 medium, > 2.5 good) and an evaluation based

on local knowledge [4].

Crayfish were investigated macroscopic and

microscopic to detect presence of the bacteria or

parasite.


Summarizing the obtained results, four crayfish

species in Latvia’s water reservoirs were identified.

Crayfish populations were determined in all similar

regions.

Noble crayfish is the dominant and native species

and it was found practically everywhere. It was

discovered in 220 from 258 locations [1, 4, 5]. It was

more distributed in Kurzeme region in opposition with

Latgale region where it was detected in five rivers

(Table 1). Approximately 36% of populations were

classified as good and medium, 32% as weak, about

others there was no information (Table 2) [1].

In 2009 the noble crayfish was found in 129 lakes

(84.9%) from all observed lakes [6]. In 2010, 1.2

thousand crayfish fry were propagates in 37 water

reservoirs [7].

The presence of narrow-clawed crayfish was firstly

reported in 1960s. Since that time the population was

expanded near Riga, Dobele, Daugavpils, Madona

regions. Narrow-clawed crayfish is widely disseminated

near Latvia—in Belaruss, Russia, Lithuania that probably

Table 1 Number of crayfish localities with the different species in different regions of Latvia [1].

Regions Noble crayfish Astacus astacus

Narrow-clawed crayfish Astacus leptodactylus

Signal crayfish Pacifastacus leniusculus

Mix populations of noble crayfish Astacus astacus and narrow-clawed crayfish Astacus leptodactylus

Total

Kurzeme 74 0 0 0 74 Latgale 63 4 0 1 68 Vidzeme 59 10 4 5 78 Zemgale 24 12 0 2 38 Total 220 26 4 8 258

Table 2 Population status of crayfish species [1, 4].

Crayfish species Weak population Medium population Good population No information Total

Noble crayfish Astacus astacus 32% 16% 20% 32% 100 Narrow-clawed crayfish Astacus leptodactylus

53% 3% 15% 29% 100

Signal crayfish Pacifastacus leniusculus 0% 0% 25% 75% 100

Preliminary Results of Crayfish Distribution and Diseases in Latvia

1144

is reason to arrive single individuals in the present

water bodies. In 2004 it was found in 34 localities and

eight of them were mixed with noble crayfish

population (Table 1) [1]. Most of populations (53%)

were classified as weak, only 18% were classified as

good and medium (Table 2). To compare in 2009

narrow-clawed crayfish was found in 25 lakes (16.4%)

from all lakes [6].

The North American signal crayfish was introduced

from Lithuania in 1971 in one locality in the Brasla

river, and in 1983 it was implemented in one lake in

Limbazi district and till 2004 it got around in another

3 localities that was far enough from the lake it was

introduced (Table 1) and only one population was

detected as good (Table 2). It means that P. leniusculus

has spread by the help of man but not in a natural way.

The signal crayfish was abundant in the lake which

was introduced in Ref. [1]. This population was

introduced due to resistance against crayfish plague

and other diseases, but it turned out that they were

very aggressive to native crayfish species and could

eject from their place. It is one of the reasons why

signal crayfish population is so widely distributed and

it serves as a disease carrier. In 2009 the North

American signal crayfish was found in one location [6].

In 2006 one more species-spiny-cheek crayfish was

registered in the Lielupe river basin. The population of

spiny-cheek crayfish is widely disseminated in

Belaruss, Lithuania and in other European countries. It

is resistant against crayfish plague and aggressive

against other crayfish species. In 2009 the spiny-cheek

crayfish were found in one location [6].

Totally the state of crayfish populations is assessed

as satisfactory. Macroscopical and microscopical

examinations showed on single individuals were

observed burn spot disease signs on crayfish, on some

individuals leech, and on some-morphological

changes. But there is no regular disease monitoring.

4. Conclusion

Four crayfish species in water bodies were detected,

and the noble crayfish is the native species.

There were prepared arrangements for restocking of

noble crayfish population in lakes.

The main threat to noble crayfish is crayfish plague

disease that eliminated the whole population. It is

necessary to detect the presence of the parasite A.

astaci in North American crayfish species—signal

crayfish and spiny-cheek crayfish to protect the

indigenous species in Latvia. It is necessary to manage

the disease monitoring measures for crayfish farming.

Acknowledgments

The author thanks Prof. Augusts Arens for the help

in providing with materials for the manuscript.

This work has been supported by the European

Social Fund within the Project “Support for the

Implementation of Doctoral Studies at Daugavpils

University” Agreement No. 2009/0140/1DP/

1.1.2.1.2/09/IPIA/VIAA/015.

References

[1] A. Arens, T. Taugbøl, Status of freshwater crayfish in

Latvia, Bulletion France Pêche Piscic (376-377) (2005)

519-528.

[2] A. Arens, Crayfish situation in Latvia and the Latvian

crayfish program, in: T. Taugbøl (Ed.), Report from the

Nordic-Baltic Workshop on Crayfish Research and

Management, Eastern Norway Research Institute and

Estonian Ministry of Environment, Fishery Department

ØF-Report 26, 1998.

[3] L. Aquiloni, M.P. Martín, F. Gherardii, J.

Diéguez-uribeondo, The North American crayfish

Procambarus clarkii is the carrier of the oomycete

Aphanomyces astaci in Italy, Biological Invasions 13

(2011) 359-367.

[4] A. Arens, Freshwater Crayfish in Latvia, Final Report,

Norway Research Institute, Lillehamer, Norway,

Latvian Crayfish and Fish Farmers Association, Riga,

Latvia, 2004, p. 52. (in Latvian)

[5] Briede, Crayfish in Latvia, Acta Biologica Universitatis

Daugavpilensis 11 (1) (2011) 83-87.

[6] Birzaks, Report on Workshop, Project LV0045

PROMIWA, unpublished data.

[7] M. Vitins, The angling cards as a contribution in

development of angling, Latvian Fisheries Yearbook

(2011) 130-132. (in Latvian)


The Effects of Simultaneous Application of Different

Organic and Biological Fertilizers on Quantitative and

Qualitative Characteristics of Cucurbita pepo L.

Mohsen Jahan, Alireza Koocheki, Mohammad-Kazem Tahami, Mohammad-Behzad Amiri and Mahdi

Nassiri-Mahallati

Agronomy and Plant Breeding Department, Agroecology Division, Faculty of Agriculture, Ferdowsi University of Mashhad (FUM),

Mashhad, Iran

Received: December 28, 2011 / Accepted: March 07, 2012 / Published: October 30, 2012.

Abstract: Understanding the relations and interactions between ecosystem components and plants is crucial for sustainable production of medicinal plants. To study the effect of simultaneous application of organic and biological fertilizers on yield and yield components of zucchini squash, split plot arrangement of factors based on randomized complete block design with three replications were used during 2009-2010 growing season. The mainplot factors were the type of organic fertilizers, including: (1) cow manure; (2) sheep manure; (3) chicken manure; (4) vermicompost; and (5) control. The subplot factors were the biofertilizes (Nitragin, containing Azotobacter sp., Azospirillum sp. and Pseudomonas sp.) utilization. The results showed the positive but non-significant effect of organic and biological fertilizers on yield and yield components of zucchini squash. Amongst the organic fertilizers, cow and chicken manure, have superiority compared to others. The highest seed oil and protein percent was obtained with application of chicken manure, however there was no significant difference between treatments in seed oil percent. The positive effect of organic and biological fertilizers on seed yield was higher than fruit yield. At a glance, application of cow manure solely was better than its application with nitragin. Nitragin application has no significant effect on some traits when utilized with sheep manure and vermicompost. Key words: Cucurbita pepo L., organic fertilizers, nitragin, growth characteristics.

1. Introduction

The cropping of medicinal plants could positively

contribute to the income of organic farms as the

guidelines for good agricultural practice for medicinal

and spice plants demands products which are not

contaminated by chemicals [1]. The requirements with

view to homogeneity and quality, particularly the

content of bioactive components are continuously

increasing so that adequate crop-specific growth

conditions need to be elaborated. Growth conditions

such as temperature, light intensity and species [2],

Corresponding author: Mohsen Jahan, Ph.D., assistant

professor, research fields: biofertilizers and organic farming. E-mail: [email protected].

nutritional factors like nitrogen, phosphorus and sulfur

supply, influencing the content of bio-active

components [3] and research needs to be carried out

for fertilizer recommendations in organic farming

which meet market requirements [4]. On the other

hand, understanding of relations and interactions

between ecosystem’s components and plants is one of

the main conditions for sustainable production of

medicinal plants.

In recent years, cultivation of medicinal plants and

other food plants with medicinal properties have been

expanded. Cucurbita pepo is an important oil seed

plant which is used in food and also in cosmetics and

health items [5]. Murkovich et al. [6] worked on

The Effects of Simultaneous Application of Different Organic and Biological Fertilizers on Quantitative and Qualitative Characteristics of Cucurbita pepo L.

1146

hundred lines of this species and found 39.5%-56.5%

oil and 21%-67.4% linoleic acid content.

The new utilizations and progressive of C. pepo in

recent years [7], call for the more researches,

especially in low input systems. Due to lack of

information about simultaneous application of

different organic and biological fertilizers on healthy

production of Cucurbita pepo L. in a low input system,

the project studies were carried out.


An experiment based on randomised complete block

design with split plot arrangement and three

replications were conducted in Research Farm of

Ferdowsi University of Mashhad in 2009-2010

growing season. Five organic fertilizers including (1)

cow manure, (2) sheep manure, (3) chicken manure,

(4) vermicompost and (5) control were allocated to

main plots and application and no application of

biofertilizer (Nitragin, CFU = 108 C/mL, containing

Azotobacter sp. and Azospirillum sp.) were assigned to

subplots. Planting was carried out on May 8, 2009 on

rows 3 m apart with 50 cm between each plant on

rows. Just before planting, the seeds inoculated with

nitragin, respecting standard conditions (particularly

avoiding of direct sunlight) and recommendations of

producer company. No chemical fertilizer or biocide

was applied and weed was controlled by hand.

Irrigation was carried out on the weekly bases. The

soil type of the experimental field was sandy clay

loam with a pH of 7.4-7.7 and 0.25%-0.30% organic

matter. Soil analysis showed 0.057 % of total N, 15

ppm and 119 ppm for available P and K, respectively.

The main nutrient elements contents of organic

fertilizers used have showed in Table 1. According to

squash nutrients requirement and manures and soil

analysis results, the amounts of 30, 30, 25 and 10 t/ha

of cow manure, sheep manure, chicken manure and

vermicompost were used, respectively.

Every 15 days after flowering stage toward, SPAD

readings were recorded with SPAD-502 DL,

MINOLTA. In the mid of flowering stage, relative

water content of leaves (RWC) were measured. At the

physiological maturity stages, the fruits were

harvested and fruit yield, seed yield, fruits number per

m2, seeds number per m2, seed weight and seed oil

and protein content were determined. The oil and

protein content of the seeds were determined using the

AOAC Official Method 972.28 (41.1.22) and 968.06,

respectively [8]. In order to approved the data have

normally distribution, a normality test was carried out,

then data were analyzed by analysis of variance

(ANOVA) and regression using Minitab statistical

software Ver. 14 and means were compared using

Duncan’s multiple range test at 0.05 probability level.


3.1 Organic Fertilizers

Chicken manure application had superiority over to

other organic fertilizers due to fruit and seed yield,

fruit and seed number per m2, individual fruit weight,

seed weight per fruit, RWC, protein and seed oil

content and SPAD readings, although this effect was

not significant (Table 2). Physiochemical

modifications occurred in soil by chicken manure was

more compared to other organic fertilizers and it may

be to high N and P content of chicken manure (Table

1). In addition, nutrients released from manure were

available for plants at the squash rapid growth stage,

Table 1 Nutrient contents of different organic fertilizers used in the experiment.

N (%) P (%) K (%)

Cow manure 1.18 0.29 1.04

Sheep manure 1.21 0.47 0.92

Chicken manure 2.14 2.35 0.78

Vermicompost 1.63 1.53 0.96


1147

Table 2 Means comparison of the effects of organic and biological fertilizers on some traits of Cucurbita pepo.

Fruit yield (t/ha)

Seed yield (g/m2)

Fruit number per m2

Seed number per m2

Single fruit weight (kg)

Seed weight per fruit (g)

1000 seed weight (g)

RWC (%)

Seed oil content (%)

Seed protein content (%)

SPAD reading

Cow manure 6.0a 16.0a 0.42a 117a 1.1a 28.6a 105a 73.9a 35.1a 29.2d 46.2a

Sheep manure 2.9a 11.8a 0.25a 48a 1.15a 25.9a 102a 70.5a 31.6a 39.1c 46.2a

Chicken manure 8.9a 16.7a 0.50a 125a 1.6a 38.2a 132a 74.9a 38.0a 41.5a 46.4a

Vermicompost 5.8a 9.0a 0.29a 65a 1.5a 23.0a 108a 68.6a 35.2a 24.6a 45.5a

Control 8.1a 14.4a 0.44a 119a 1.7a 32.9a 121a 71.0a 35.0a 40.4b 45.2a

Nitragin 5.8a 14.3a 0.37a 91a 1.3a 30.7a 108a 69.7a 35.4a 32.4b 45.7a

No Nitragin 6.8a 12.8a 0.39a 97a 1.5a 28.9a 119a 73.9a 34.8a 37.5a 46.1a

For each factor and in each column, the values which followed by the same letter, are not significantly different at 5% level.

thus, nutrients leaching reduced to a minimum level

[9-11].

In each column, means followed by the same letters

have not significantly difference (P < 0.05).

As it was shown on Fig. 1, SPAD readings were

high in all organic fertilizers compared to control at

the end of growing season. There are evidences that

indicating cattle manure application increased leaf

chlorophyll content and growth of some crops [18].

3.2 Biofertilizers

Nitragin inoculation resulted in the higher seed

yield, seed weight per fruit and seed oil content rather

than no nitragin application, although there was no

significant difference between application and no

application of nitragin due to these traits (Table 2).

There are evidences indicating the positive and

promotional effects of rhizobacteria on plant growth

and development [12, 13]. These effects assigned to

microbial activities in synthesis of phytohormones,

organic acids and vitamins, nitrogen fixing, increased

some nutrients availability like phosphorus and finally

interactions between PGPRs and other soil

microorganisms in the rhizosphere which benefits the

plant growth [12, 14, 15].

3.3 Interaction of Organic Fertilizers and

Biofertilizers

The positive effect of nitragin on seed yield was

more revealed than fruit yield (data not shown). As it

was shown in Fig. 2, nitragin inoculation resulted in

higher seed yield when combined with sheep manure,

vermicompost and in control treatment, however,

when nitragin combined with cow and chicken manure,

Fig. 1 C. pepo SPAD readings trend through growing season resulted from application of different organic fertilizers.


1148

8.07

17.0115.73

12.58

16.02

21.3

6.64

17.79

5.51

12.94

0

5

10

15

20

25

Cow Sheep Chicken Vermicompost Control

Organic fertilizers

See

d y

ield

(g.

m2 )

Nitragin

No Nitragin

Fig. 2 Interaction of different organic and biological fertilizers on seed yield of Cucurbita pepo.

34.37

22.32

35.63

43.83

26.97

45.35

41.52

28.42

41.66

30.05

10

15

20

25

30

35

40

45

50

Cow Sheep Chicken Vermicompost Control

Organic fertilizers

See

d o

il co

nte

nt

(%)

Nitragin

No N itragin

Fig. 3 Interaction of different organic and biological fertilizers on seed oil content of Cucurbita pepo.

it seems that positive effect was not appeared, even

though, there was no significant difference between

seed yield resulted in chicken manure with and

without nitragin inoculation.

Interaction of organic and biological fertilizers, on

seed weight, fruit and seed number per area unit, and

single fruit weight were similar and have superiority

on control (data not shown).

It was reported that combined utilization of

vermicompost and compost, with Pseudomonas and

Azotobacter, increased fennel yield significantly

compared to solely utilization of compost,

vermicompost, Pseudomonas and control [16]. Other

researches have emphasised on positive effects of

vermicompost on soil and plants characteristics [10,

17].

The highest seed oil percent resulted from chicken

manure without nitragin inoculation and the lowest

amount observed in plants treated with sheep manure

plus nitragin inoculation (Fig. 3).

Interaction of organic and biological fertilizers was

resulted in significant difference amongst treatments

regarding to seed protein content (data not shown).

4. Conclusions

In general, the results showed amongst organic

fertilizers used in this experiment, the chicken manure

solely or combined with nitragin, has superiority


1149

compared to other organic fertilizers, although, chicken

and sheep manure, and vermicompost application in

combination with or without nitragin inoculation, were

not resulted in significant differences due to most

studied traits. Cow manure solely application was better

than in combination with nitragin.

At a glance, utilization of biofertilizer combined

with organic fertilizers could be resulted to an

optimum quantitative and qualitative yield without

any agrochemicals in a low input production system

of zucchini squash.

References

[1] Europam, Leitlinien für die gute landwirtschaftliche praxis (GAP) von arznei- und gewürzpflanzen, Z Arznei- & Gewürzpfl 3 (1998) 166-174.

[2] E.A.S. Rosa, P.M.F. Rodrigues, The effect of light and temperature on glucosinolate concentration in the leaves and roots of cabbage seedlings, J. Sci. Food Agric. 78 (1998) 208-212.

[3] E. Bloem, S. Haneklaus, E. Schnug, Significance of the sulfur nutrition for the pharmaceutical quality of medicinal plants, in: Proc. 12th World Fertilizer Congress, Beijing, China, 2001a.

[4] P. Griffe, S. Metha, D. Shankar, Organic Production of Medicinal, Aromatic and Dye-Yielding Plants (MADPs): Forward, Preface and Introduction, FAO, Rome, Italy, 2003.

[5] E. Bombardelli, P. Morazzoni, Cucurbita pepo L., Fitoterapia LXVIII (4) (1997) 291-302.

[6] M. Murkovic, A. Hillebrand, H. Winker, W. Pfannhauser,

Variability of vitamin E content in pumpkin seeds

(Cucurbita pepo L.), Z. Lebensm Unters. Forsch. 202

(1996) 275-278.

[7] P. Schinas, G. Karavalakis, C. Davaris, G. Anastopoulos,

D. Karonis, F. Zannikos, et al., Pumpkin (Cucurbita pepo

L.) seed oil as an alternative feed stock for the production

of biodiesel in Greece, Biomass and Bioenergy 33 (2009)

44-49.

[8] AOAC, Official Methods of Analysis, 18th ed., Washington DC, Assoc. Off. Anal. Chem., 2005.

[9] J.O. Azeez, A.B. Van Averbeke, W. Okorogbona, Differential responses in yield of pumpkin (Cucurbita maxima L.) and nightshade (Solanum retroflexum Dun.) to the application of three animal manures, Bioresource Technology 101 (2010) 2499-2505.

[10] K.S. Arun, A Handbook of Organic Farming Pub., Agrobios, India, 2002.

[11] A. Robin, R.A.K. Szmidt, W. Dickson, Use of Compost in Agriculture, Frequently Asked Questions (FAQs), Remade, Scotland, 2001, pp. 324-336.

[12] J.M. Barea, M.J. Pozo, R. Azcon, C. Azcon-Aguilar, Microbial co-operation in the rhizosphere, Journal of Experimental Botany 56 (2005) 1761-1778.

[13] I.R. Kennedy, A.T.M.A. Choudhury, M.L. Kecskes,

Non-symbiotic bacterial diazotrophs in crop-farming

systems: Can their potential for plant growth promotion

be better exploited?, Soil Biology and Biochemistry 36

(2004) 1229-1244.

[14] J. Chen, The combined use of chemical and organic

fertilizers and/or biofertilizer for crop growth and soil

fertility, in: International Workshop on Sustained

Management of the Soil-Rhizosphere System for

Efficient Crop Production and Fertilizer Use, Thailand,

Oct. 16-20, 2006.

[15] J.K. Vessey, Plant growth promoting rhizobacteria as biofertilizers, Plant and Soil 255 (2003) 571-586.

[16] R. Moradi, P. Rezvani Moghaddam, M. Nassiri, A.

Lakziyan, Study the effects of biological and organic

fertilizers on yield, yield components and essence amount

of fennel (Foeniculum vulgare), Iranian Journal of Field

Crops Research. 7 (2010) 625-635. (in Persian with

English abstract).

[17] N.Q. Arancon, C.A. Edwards, P. Bierman, C. Welch, J.D.

Metzeger, Influences of vermicomposts on field

strawberries: 1. Effects on growth and yields, Bioresource

Technology 93 (2004) 145-153

[18] M. Jahan, A. Koocheki, M. Nassiri-Mahallati, Effects of

arbuscular mycorrhizal fungus and free living nitrogen

fixing bacteria on growth characteristics of corn (Zea

mays L.) under organic and conventional cropping

systems, in: 2nd Scientific ISOFAR Conference

“Cultivating the Future based on Science”, Modena, Italy,

June 18-20, 2008.


The Impact of Deforestation in Anambra State: The

Ekwusigo Example

Joel Ekwutosi Umeuduji and Chukwuma Onyebueke Egbuonu

Department of Geography & Environmental Management, University of Port Harcourt, Port Harcourt 5323, Nigeria

Received: March 21, 2012 / Accepted: July 12, 2012 / Published: October 30, 2012.

Abstracts: It is a fact that demographic and socio-economic developments are exploitatively exerting severe pressure on forest resources in Nigeria. Not only has the economy of the people been severely affected but also the environment has witnessed accelerated soil erosion, especially in Eastern Nigeria. Accordingly, this study set out to explore the environmental and socio-economic impacts of deforestation using an empirical case. Two forested and two deforested sites in Ekwusigo L.G.A of Anambra State were closely studied with respect to deforestation indices. From the data generated, Student’s t-test was used to attempt a statistical comparison of the forested and deforested sites. The findings indicate that forest cover depletion affected both the canopy openness and the number of non-timber forest products in the area. Finally, the paper stressed the need to maintain a sustainable plant cover while economically harnessing forest resources. Key words: Deforestation, plant cover, canopy openness, non-timber products, economical harnessing, environment.

1. Introduction

Occasionally, natural events such as lightning and

other extreme catastrophes relating to climate change

have led to deforestation. However, globally and more

commonly, human activities especially in the form of

agriculture, urbanization and industrialization have

become the major reasons for extensive removal of

plant cover. At the beginning of the 21st Century,

there were approximately 3.5 million hectares of

forests in the world, out of which 2 million hectares

were in the developing countries, mostly in the

tropical and sub-tropical regions [1].

Tropical deforestation, especially in the rainforests

is now widely recognized as one of the most critical

environmental problems facing the world today with

serious long term economic and social consequences

[2].

A close look at man/plant cover relationship shows

Corresponding author: Joel Ekwutosi Umeuduji, Ph.D.,

research field: physical geography (geomorphology). E-mail: [email protected].

that deforestation is a necessary trade-off for human

survival. Frey (2002) built a regression model using a

broad spectrum of independent variables including

family and farm characteristics as well as agricultural

inputs so as to highlight the factors that drive farmers

to deforest the land in Rondonia, Brazil [3]. Similarly,

Shandra examined the determinants of deforestation

for up to 73 nations from 1990 to 2000 noting that

economic growth and population growth significantly

accentuate the process [4].

Both directly and indirectly, deforestation has been

causally linked to several other environmental issues

that threaten human survival. Using the Brazilian

Amazon as a case, Fearnside and Laurance argued

with empirical evidence that the effect of the

1990-1997 deforestation in the Amazon region gave a

notable impetus to carbon emission and global

warming [5]. In Indonesia, Warr and Yusuf used a

general equilibrium model to examine the

effectiveness of subsidy to encourage forestry land use

with a view to achieving the target of reducing

deforestation-based green house gas emissions by

The Impact of Deforestation in Anambra State: The Ekwusigo Example

1151

26% in 2020 [6].

The process of deforestation essentially gives rise to

environmental conditions that are adverse and quite

insidious to human wellbeing and survival. For

instance, it has been argued that empirical researches

have shown that the risk of being bitten by the

malaria-carrying mosquito is nearly 300 times higher

in cleared areas than in those that are largely

undisturbed [2]. According to them, clearing forest for

cropland has created more breeding sites for the

Amazon basin’s most efficient malaria-carrying

mosquito, Anopheles darlingi, which prefers to lay its

eggs in deep waters surrounded by short vegetation.

With 3.12% per annum as its rate of forest

conversion, and having lost 55.7% of its primary

forest between 2000 and 2005, Nigeria had the

“discredit” of having the highest rate of deforestation

worldwide [7, 8]. In Akwa Ibom, Anambra, Cross

River, Delta, Edo, Ondo and Rivers states, forest

resources are so rapidly being exploited that savannas

and grasslands might soon develop in these typically

rainforest areas.

In Ekwusigo L.G.A of Anambra state with a

population density exceeding 400 person/km2,

subsistent farming, hunting, craft making, fuel wood

collection and moderate logging are major economic

activities of the people. In this area, as in most other

parts of the country, deforestation studies have put

much emphasis on the socio-economic and ecological

impacts and benefits derivable from the practice.

However, it is clear from existing literature that issues

relating to the best methods of altering vegetal cover

as well as the health implications of such alterations

have not been fully addressed. Accordingly, the major

task facing this investigation is to identify the most

preferred method of deforestation or plant cover

alteration by the people as well as its suitability to the

environment of the area in question. Again, it is

obvious that deforestation clearly depletes timber

reserves but the effect of this process on non-timber

forest products has largely remained less emphasized.

Also, assessing the impact of deforestation on canopy

openness and its implication on the environment has

remained a critical issue that requires explanation.


Following a detailed reconnaissance [9], a survey

design was adopted to take stratified samples from

deforested areas and compare with non-deforested

areas. Purposively, two major towns or settlements

(Ozubulu and Oraifite) were sampled from the

deforested areas and two other settlements (Ichi and

Ihembosi) from forested areas (Figs. 1 and 2). For data

collection on key deforestation parameters (such as

canopy openness, number of trees felled and number

of non-timber products destroyed), each of the four

sample sites was divided into plots of 50 m × 100 m.

Twenty plots each were sampled from deforested and

forested areas. Also, close-ended questionnaires were

administered to 20 randomly selected family heads

from each of the four towns.


Tables 1 and 2 show a detailed inventory of mature

trees of > 25cm dbh (diameter at breast height) on the

forested and deforested plots. From a cursory

comparison of the total values (column 5) in the two

tables, there appears to be more damaged and

destroyed trees from the deforested plots than those

from the forested plots. The different forms of damage

(minimal, moderate and severe) are due to different

forms of human activities which give rise to

deforestation. To establish that the depletion of mature

trees in the deforested plots was a function of

deforestation, the Student’s t-statistic was applied to

the data sets. At 95% probability level and 38 degrees

of freedom, the calculated t value was 28.54 and well

above the critical t value of 2.02, showing a glaring

difference in the two data sets and deforestation

statistically accounts for this difference.

Though logging for timber products is a noticeable

drive for deforestation, yet it should be noted that a


1152

Fig. 1 Anambra State: showing Ekwusigo L.G.A..

good number of non-timber forest products are

inevitably and contemporaneously lost in the process.

These range from oil palm, raffia palm, bamboo,

pawpaw, banana/plantain trees to creeping and

climbing plants. From the forested plots, the palms

were mostly affected in the process of fruit

harvesting and palm wine tapping. Other vulnerable

plants such as pawpaw as well as climbing and

creeping plants are easily destroyed by hunters who

cut their way through the forest. On the deforested

plots, these same vulnerable non-timber plants are

not only destroyed in the process of logging, but also

through clearing for farm lands and building

construction.

Table 3 shows a census of such non-timber forest

products severely affected by human activities and

remarkably, the difference in the totals is quite wide.

Again, at 95% probability level and 38 degrees of


1153

Fig. 2 Ekwusigo LGA: showing the sampled settlements.

freedom, a calculated t value of 6.61 (and 2.02 as

critical), it is clear that the difference in the two sets of

data is statistically significant. Our survey using

questionnaire shows that as much as 46.25% of the

respondents believe that deforestation in the area can

adversely affect the environment.

As shown in Table 4 and Fig. 3, a good number of

the respondents (82.5%) apparently prefer and use

cutlass, axe and other mild cutting implements to clear

the forest as opposed to more damaging methods such

as the use of chain saw, graders or fire. This, to an

extent indicates that the people are interested in

preserving their environment and protecting the soil

from erosion.

4. Discussion

Goodland and Irwin [1] built a model that captures

the numerous effects of forest disturbance on the

general ecosystem of a place (Fig. 4). The model

summarizes almost all the consequences of logging


1154

Table 1 Damaged trees of > 25 cm dbh on deforested sites.

Plots Damage Type

Severe (Uprooted or cut) Moderate (Broken branches) Minimal form of bark damage Total

1 4 15 4 23

2 5 21 4 30

3 6 16 3 25

4 7 14 1 22

5 10 10 1 21

6 6 12 2 20

7 6 8 4 18

8 7 7 3 17

9 11 5 1 17

10 9 10 8 27

11 7 12 2 21

12 8 10 2 20

13 4 13 5 22

14 3 9 10 22

15 2 10 8 20

16 2 12 7 21

17 1 8 5 14

18 6 7 6 19

19 5 9 5 19

20 3 5 1 9

Source: Authors’ field work, August, 2010.

Table 2 Damaged trees of > 25 cm dbh on forested sites.

Plots Damage Type

Severe (Uprooted or cut) Moderate (Broken branches) Minimal (Any form of damage) Total

1 2 7 11 20

2 1 13 2 16

3 2 3 3 8

4 - 4 - 4

5 - 8 1 9

6 2 4 1 7

7 1 2 2 5

8 1 2 1 4

9 1 12 3 16

10 - 7 6 13

11 3 8 3 14

12 - 10 3 13

13 1 6 2 9

14 2 14 3 19

15 2 3 4 9

16 1 4 12 17

17 - 3 11 14

18 4 4 1 9

19 2 2 5 9

20 - 2 1 3



1155

Table 3 Number of non-timber forest products damaged/destroyed on forested and deforested areas.

Plots Number damaged/destroyed in Forested Aras Number damaged/destroyed in Deforested Aras 1 12 13 2 7 10 3 9 12 4 8 7 5 8 9 6 14 12 7 5 8 8 4 8 9 6 10

10 8 12 11 7 6 12 3 17 13 11 16 14 7 9 15 6 4 16 12 6 17 7 6 18 8 8 19 9 12 20 3 5

Total 154 190


Table 4 Methods of forest clearing in Ekwusigo.

Method Response Percentage

Manual Cutting 66 82.50

Use of Fire 7 8.75

Graders 3 3.75

Chain Saw 4 5.00

Total 100 100.00


Fig. 3 Methods of forest clearing in Ekwusigo.


1156

LOGGING AND FOREST DISTURBANCE

Open Bush Decreased

Evapotranspiration Reduced Shade

Increased insolation

Induration

Erosion

Flood

Siltation

Soil

desiccation

Low Humidity

Harsh Micro‐climate

Disturbed Mineral Cycling

Reduced rainfall

Drought

Retarded

Regeneration

Species Depletion

Decreased

Water

Loss of

NTFPs

Less plant

growth

Fig. 4 Model of the effects of forest disturbance on the ecosystem. (Modified based on Goodland and Irwin [1]).

and forest depletion in the ecosystem. This model,

which was obviously synthesized from different

literature-based conclusions, is not only a veritable

backdrop but also a solid stepping stone for empirical

investigations.

Canopy openness refers to gaps in forest canopies

created by events of forest destruction which could be

natural or man-made.

Mature, broad-leaved trees with widespread

branches provide larger canopies. However,

deforestation and other related activities always lead

to the reduction or removal of these canopies. In line

with the Goodland and Irwin deforestation model,

we have statistically established that canopy

openness was as a result of deforestation and that is

why it is more pronounced in the deforested section

of our study area. We have also empirically

established that deforestation leads to a noticeable

level of species depletion and loss of non-timber

forest products as confirmed by respondents who

admitted that deforestation adversely impacts on the

environment.

5. Conclusion

We have attempted exploring the impact of

deforestation on the environment by highlighting

some indices of canopy openness and the possible

effects on non-timber products. Empirically, the

impacts were found to be more in the deforested sites

as opposed to the forested sites. The impact of

deforestation on the environment obviously appears to

be a major driver of soil erosion in Ekwusigo area.

This is because, given the already weak

geological/lithological setting, the indices examined

significantly expose the area to the onslaught of

erosion re-enforced by some topographical and

climatic factors.

Not only does deforestation affect the environment,

but also it has serious implications on the economy of

the people. Ironically, in Ekwusigo, deforestation is

re-enforced by people’s subsistent economic survival

practices, especially the quest for agricultural land on

which to grow food crops for survival. This, in many

cases, is done on gentle slopes where runoff easily

concentrates, initiates and accelerates soil erosion.


1157

What is therefore basically required in this region is to

create an awareness of the environmental implications

of man’s agricultural activities as well as the need to

strike a balance between man’s survival strategies and

environmental sustainability.

References

[1] Chatham House, Illegal Logging, Mongabay.com, 2011, http://www.illegal-logging.info/approach.php.

[2] R.S. de Fries, R.A. Houghton, M.C. Hansen, Carbon emission from tropical deforestation and re-growth based on satellite observations for the 1980s and 1990s, National Academy of Science (99) (2002) 1456-1469.

[3] C.O. Egbuonu, Effects of Deforestation on the Prevalence of Malaria in Ekwusigo L.G.A, Anambra State, Unpublished M.Sc. Thesis, University of Port Harcourt, 2011.

[4] FAO, Deforestation in Nigeria, 2005, http://en.wikipedia.Org/wiki/deorestationinNigeria.

[5] P.M. Fearnside, and W.F. Laurance, Tropical deforestation and greenhouse-gas emissions, Ecological Applications 14 (2004) 982-986.

[6] E.F. Frey, Tropical deforestation in the Amazon: An

economic analysis of Rondonia, Brazil, Political

Economy 11 (2002) 1-7.

[7] R.J.A. Goodland, H.S. Irwin, Amazon Jungle: Green Hell

to Red Desert?, Elsevier Scientific Publication,

Amsterdam, 1975.

[8] J.M. Shandra, The world polity and deforestation,

International Journal of Comparative Sociology 48 (1)

(2007) 5-27.

[9] A.Y. Vittor, R.H. Gilman, J. Tielsh, The effect of

deforestation on the human-biting rate of Anopheles

darling—the primary vector of Falciparum malaria

in the Peruvian Amazon, American Journal of

Tropical Medicine and Hygiene 74 (2006) 3-11.

[10] P. Warr, A.A. Yusuf, Reducing Indonesia’s

deforestation-based greenhouse gas emissions, Australian

Journal of Agricultural and Resource Economics 55

(2011) 297-321.


Contribution of Study Bioecology of the Fauna

Chamaerops humilis in the Region of Tlemcen (Algeria)

Damerdji Amina

Department of Ecology and Environment, Faculty of S.N.V/S.T.U, University of Tlemcen, Tlemcen 13000, Algeria

Received: February 02, 2012 / Accepted: July 12, 2012 / Published: October 30, 2012.

Abstract: The region of Tlemcen is situated in the north-west of Algeria. The aridity of the climate had lead to the development of the matorral, a state of degradation of the Mediterranean, and the composed xerophytes plants such as doum and diss, had been found. Chamaerops humilis, xerophyte plant, with special morphologic and botanic character presents a resistance of these climatic. The authors have proposed study of fauna closly linked to this plant. A faunistic inventory was realized in the Mansourah area (region of Tlemcen). Four stations have been described. Collecting sample was performed during June 2003-Mar. 2004, replying on sixteen (16) prelevements. The number of species were estimated of about 136, in which 111 are Arthropoda, the Entomofauna represented by 97 species and the other inventory are Arachnida by 8 species and Myriapoda by 6 species. 18 species are related to Gastropoda. The vertebrates are few. The importance of different groups’ recolted on the Chamaerops humilis in the four stations is done particular to the insects. Analysis factorial correspondence (A.F.C) show different grouping of animal species. Key words: Chamaerops humilis, fauna, inventory, bioecology, region of Tlemcen (Algeria).

1. Introduction

The objective of this study is to understand the

faunal diversity of this plant drought-tolerant on one

hand and to detect characteristics of species

subservient on the other. The authors consider it

useful to compare the results with other plants

adapting to the same conditions.

Little work has been done on wildlife Chamaerops,

the associated malacofaune Chamaerops humilis [1-3],

the insect fauna [4-7] and invertebrate fauna [8].

A study of wildlife bioecological subservient to

doum palm, also known as dwarf, is carried out in

four stations Mansourah zone in the region of

Tlemcen. Before discussing the results, it is necessary

to describe the host plant and give the methodology of

work.

The results focus first on the inventory of the

different animal species encountered on this plant

Corresponding author: Damerdji Amina, Dr, M.C.A., research fields: malacology, entomology and ecology. E-mail: [email protected].

xerophyte then on a comparison between four stations

for different groups of fauna, focusing on insects.

Finally, factor analysis of correspondence is

discussed.


2.1 Choice of Plant Material

Plante shrub is often called palm-dwarf exceeding 1 m

tall (Fig.1). It generally grows to clump. The root is

deep and pivoting. The trunk or stipe is often short,

bulbiforme. The leaves are persistent grouped at the

top of stipe. They are in the form of segments

lanceolated. The inflorescences are generally

composed spadices.

The fruit is a yellowish or reddish bay of variable

size.

The systematic of this plant species is:

Embranchement spermaphytes under

embranchement angiosperms;

Class monocotyledons;

Contribution of Study Bioecology of the Fauna Chamaerops humilis in the Region of Tlemcen (Algeria)

1159

Order palmales (spadiciflores);

Family palmaceae;

Genre chamaerops;

Genre species Chamaerops humilis subsp

argentea;

Name vulgar palm-dwarf;

Arabic name doom.

The doum is a kind of Monocotyledone in the

Mediterranean region. It grows to the state in

spontaneous matorrals. It prefers limestone substrates.

It meets next stages bioclimatic at various altitudes.

This species is rich in plant materials carbohydrate, fat

and alkaloids. In addition, its high ornamental value,

Fig. 1 Morphology of clumps’ Chamaerops humilis.

doum presents utilitarian aspects in the manufacture of

baskets, hats, ropes and shoes.

2.2 Choice Stations Study

To accomplish this work, the authors prospected

four stations in the area Mansourah (Fig. 2). The

chosen stations are described in Table 1.

The transects plants are made for each station to

determine the approximate percentage of recovery by

this plant.

Tlemcen taken as a reference station between 1998

and 1999 lies in the floor bioclimatic semi-arid

temperate winter.

2.3 Methodology

The experimental protocol produced for the four

stations is the same.

The samplings conducted from are distributed June

2003 and March 2004 in 16 samples. It is obvious that

we use methods easy to implement but provide

sufficient results. The shells of molluscs are harvested

by hand. Regarding insects and according to their

mode of locomotion (theft, walking…), we use

traditional hunting methods (insect nets…) and

trapping methods (insect - Myriapodes - Arachnida)

Pots-traps are placed at the foot of a tuft at its.

Fig. 2a Geographical situation for stations of Chamaerops humilis in the zone of Tlemcen.


1160

Station (1) Station (2)

Station (3) Station (4)

Fig. 2b Photos station of Chamaerops humilis.

Table 1 Data on soil and botanical 4 stations prospecting.

Stations studied Slope, % Altitude, m Humidity, % Nature of soil CaCO3, % Recovery of Chamaerops, %

Station (1) 5-10 720 7 Limono-clay 0.57-1.05 15-25

Station (2) 15-20 750 3.6 Limono-clay 1.06-1.54 20-30

Station (3) 1-5 700 10.5 Limono-clay and sandy 2.04-2.49 20-25

Station (4) 25-30 630 12.5 Limono-clay and sandy 2.60-3.83 30-40

periphery and between two clumps. The yellow color

remains attractive for the majority of insects Pterygota

(Hymenoptera-Hemiptera).

2.3.1 Methods Used for Inventory

The species caught are brought in bottles of hunting

and killed with ethyl acetate. The fragile insects are

pinned on etaloirs. We use the binocular microscope

to determine the small forms.

As far as molluscs, the determination of gastropods

were made by us and this from conchyliologiques

characters.

Regarding the other groups, various documents are

consulted for arachnids [9, 10], insects [11-13, 15] and

the vertebrates (reptiles and birds) [14, 16, 17].

2.3.2 Analysis Factorial Correspondence

The species caught may form presence-absence be

regarded as its setting, treating an array of statements

(middle + species) and analyze relations and

species-middle (station) in the form of liaison between

modalities [18].

After projection additional individuals who

revealed in the form of dispersion in the various plans

under consideration (or Gauss curves along an axis),

identify and compare the ecological niches of species

caught.


3.1 Inventory of Harvested Species of Fauna

The values of wealth specific groups of fauna are

given in the following Table 2.

In the part of Invertebrates, There are only three

classes are Vertebrates. The arthropodo fauna remains

the largest species richness with a gathering of 111


1161

arachnïdea, including myriapoda and insects.

3.2 Relative Importance of Different Groups

Harvested in the Station Doum

The relative abundances different groups of fauna

harvested in 4 stations are given in Fig. 4.

3.3 Relative Groups’ Entomofauniques in 4 Stations

Doum

The entomofauna includes 97 species of which 92

are Pterygota and only 5 are non-winged insects,

(Collembola with 3 species and Thysanoura with 2

species).

The large number of insects Pterygota is represented

by 10 orders namely beetles, wasps, lepidoptera, diptera,

orthoptera, dermaptera, mantoptera, hemiptera,

phasmidoptera and odonaptera.

3.4 Factor Analysis Correspondence (A.F.C)

The table presence-absence, has enabled us to

establish this analysis. We noted:

A contribution to the total inertia (percentage

explained by the main axes):

Axe 1 = 35.73%;

Table 2 Value of wealth specific groups of fauna harvested in the doum in 4 stations.

Groups faunistiques Number of species

Invertebrata

Gasteropoda 18

Arthropoda

Arachnoïdea 8

Myriapoda 6

Insecta

Collembola 3

Thysanoura 2

Coleoptera 22

Hymenoptera 16

Lepidoptera 19

Diptera 13

Orthoptera 14

Dermaptera 2

Mantoptera 2

Hemiptera 2

Phasmidoptera 1

Odonaptera 1

Vertebrata

Reptilia 5

Aves 1

Mamalia 1

Total 136

Fig. 3 Relative abundance of different groups of fauna harvested in stations doum.


1162

Fig .4 Relative importance of different groups of fauna harvested in 4 stations.

Fig. 5 Relative importance of different groups entomofauniques harvested in 4 stations.


1163

a

b c Fig. 6 a: Plot groups’ fauna, (axe 1-2 lines); b: Plot groups’ fauna, (axe 1-3 lines); c: Plot groups’ fauna, (axe 2-3 lines).


1164

Axe 2 = 32.66%;

Axe 3 = 31.60%;

Axe 4 = 0.

The three axes 1, 2 and 3 are sufficient for this

analysis, the settlement fauna contributes to a

relatively inertia explained for each theme:

The species contribute differently to the formation

of the axis 1. We selected:

Grouping (A)

Archelix polita punctatiana (5); Timarcha

tenebricosa (40); Pollema rudis (100) with 91.97%

for one.

Grouping (B)

Sphincterochila candidissima (1); Eobania

vermiculata (8); Euparypha pisana (9); Meloe sp. (44);

Polyommatus icarus (74); Camponotus sp. (84);

Polistes gallicus (89); sp. of Hymenoptera (not

specified) (94); Pezotettix giournai (110); Acrotylis

patruelis (12); Ameles nana (125) with 87.92% for

one.

Grouping (C)

Helicella (Cernuella) virgata (12); Helicella

(Xerovera) globuloïdea (17); Collembo podurata (35);

Lepisma saccharina (37); Otiorrhynchus sp. (50);

Lycaena phlaeas (72); Cataglyphis bicolor (82);

Sphex ingeus (87); Leptomydas corsicanus (102);

Fristalis Tenax (105) with 85.27% for one.

Grouping (D)

Machilis sp. (36); Gryllus sp. (121); Sphodromantis

lineola (124) three species with 71.20% for one.

They participate on the axis 1 and 3 in station 1 and

3 in which this group of species specific

characterization depends on these two stations

vis-à-vis climate and especially the vegetation.

Grouping (E)

Helix (Alabastrina) soluta (10); Chrysomela

americana (38); Mylabris duodecimpunctata (43);

Utethesia (Deiopeia) pulchella (63); Colias hyale (67);

Thereva plebeia (103); Pyrgomorpha conica (108);

Forficula auricularia (122); Lacerta sp. (133);

All these species listed contribute strongly in line

with a 1% greater than 50. Other species involved

lower.

This led us to consider the axis 2:

Grouping (G)

Brachycerus sp. (45); Arctia caja (60); Vanessa

atalanta (77); Rheidol pallidula (80); Vespula

germanica (90); Mus musculus (136) with 72.72% for

one.

Grouping (F)

Latrodectus sp. (24); Archelix wagneri (7);

Phrynichus reniformis (22); Sminthrus viridis (42);

Spercheus emarginatus (62); sp. not determined

(Lepidoptera) (64); Anthocharis cardammes (68);

Camponotus ligniperda (83); Vespa crabo (88);

Chamaemya aridella (104); Oedipoda coerulescens

sulfurescens (114); Sphingonotus rubescens (117);

Lygaeus militaris (127); Carausius morosus (128);

Chameleon sp. (130).

Other species involved so low with a lower

percentage to 50%.

The axis 3 also contributes by its inertia lowest

compared with other axes.

We note that 3 species of molluscs and a kind of

Coleoptera have a high percentage about 92 (group H)

or Macularia hieroglyphicula (3), Helicella

(Trochoides) cretica (15), Rumina decollata (18),

Agriotes obscurus (58). Five other species are

involved with a lower percentage slightly above 50%.

And all other species have a percentage that does not

go beyond 46%. The species of fauna by their

different characteristics and 4 stations studied

separately participate in the formation of axes 1, 2 and

3. In addition, the particular case of the station 2

shows its participation significant groupings of 3 axes.

Groupings (A), (E) and (C), 3 groups are to the

right and the group (D) to the left corresponding to

differences in microclimate and vegetation of the first

station (Complex university), It is an environment

where the rate of recovery of Chamaerops humilis

between 15% and 25%, with a rate of limestone

(CaCO3) less important (0.57% to 1.05%) is a


1165

relatively less humid. The grouping (F) with a

percentage ranging between 66.52% and 68.04%

consists of common species between the construction

of lines 1 and 2. These species are in the first station.

Species that are the groupings (B) (E) and (H) 2-3 axis

characterized the first two stations (Complex

University and the city of 400 dwellings). These last

two very close to each other on the one hand and on

the other hand, were almost the same characteristics

abiotic and biotic. The grouping D: Machilis sp. (36),

Gryllus sp. (121) and Sphodromantis lineola (124) on

the same axis 2-3, are participating this time on the

axis 3, i.e. characterize the cultivated field. The station

3 is a relatively wet on a soil that is limono-sandy clay.

The species grouping (G) on the axis 1-2 and those of

grouping (H) 1-3 axis have a special adaptation to the

fourth station (Bouhenak). It is characterized by high

humidity due to the proximity of oueds, and hence a

recovery of Doum largest and texture of the soil that is

limono-sandy clay.

On Chamaerops humilis [8] have counted 136

species while on Ampelodesma mauritanicum [19]

have counted 112 species of which 88 insects. In the

study carried out on the Rosemary biocenotic 218

animal species are found where the insect fauna ranks

first with 176 species [20]. On Calycotome spinosa,

161 species of which 131 are met with 115 insect

fauna of arthropods [21]. In the extreme west of the

Algerian coast, [22] have inventoried 131 species of

Arthropoda in which 116 are insects. The importance

of invertebrates (93 species) and particular of the

insect fauna (78 species) is rated among the total

fauna recorded in the southern area of Tlemcen

[23].On Cistus salvifolius stations, 72 species of

invertebrates have been identified [24]. It has been

counted that about fifty species in the stepp zone in

the stations Stipa tenacissima where the majority of

species are insect fauna [25]. Similar results for A.F.C

were obtained previously [1]. By ordination of species

and stations, 3 invertebrate communities are

determined and they are closely connected to the three

stations of Ampelodesma mauritanicum [19].

4. Conclusion

The total number of wildlife species harvested from

the Doum east of 136. The Arthropods are the most

important specifically. Insects alone constitute over

85% of arthropodofauna with species richness of 97.

The gastropods are represented by 18 species. The

Vertebrates (Reptiles-Birds-Mammals) are limited.

The importance of Hymenoptera for 4 stations on the

one hand and on the other hand those who come to

gastropods 2nd position with a high percentage

equivalent to 22.12% for the fourth station.

The factor analysis of correspondence (A.F.C.)

highlights several groups of animals. They are well

organized on three axes vis-à-vis the characters

edapho-climatic and botanical prospecting of 4

stations. The groupings respectively rated from A to H

give us more information for the species on the one

hand and stations on the other. We cite as an example

Machilis sp. (36), Gryllus sp. (121) and

Sphodromantis lineola (124), entomofaunical species

all three present in the station cultivated field. Should

make it a rapprochement between certain species of

fauna and Doum (Plante xerophilic) given its

botanical characteristics becoming a preferendum for

these species.

References

[1] B. Bouhellou, Contribution à l’étude bio-écologique de la faune de Chamaerops humilis L. (Doum) (Monocotylédones, Palmacées) dans la région de Tlemcen. Mém. Ing. Ecol. I.S.N. Univ. Aboubekr Belkaid- Tlemcen, 1998, p. 93.

[2] A. Damerdji, B. Bouhellou, Entomofaune du Chamaerops humilis (Doum) dans la région de Tlemcen: Inventaire–Importance saisonnière–Importance mensuelle des principaux groupes d’Insectes Ptérygotes, 3ème Journée d’Entomologie, I.N.A. El-Harrach, Alger, May 15, 1999.

[3] A. Damerdji, La malacofaune associée au Doum: Inventaire—Aperçu bioécologique dans la région de Tlemcen (Algérie), Comm. Orale, II International Congress of European Malacological Societies, Vigo, Espagne, Septembre 9-13, 2002.


1166

[4] A. Damerdji, B. Bouhellou, Inventaire des insectes recensés sur le Chamaerops humilis (Doum). Comm. affichée (Poster), Journée d’étude sur l’Entomologie, Labo. Eco. Ani., I.S.N. Univ. Aboubekr Belkaid, Tlemcen, May 09, 1998.

[5] A. Damerdji, B. Bouhellou, Inventaire de l’entomofaune du Chamaerops humilis (Doum) dans la région de Tlemcen. Comm. affichée (Poster), 2ème Journée d’Entomologie, I.N.A. El-Harrach, Alger, Mars 16, 1998.

[6] A. Damerdji, B. Bouhellou, Entomofaune du Chamaerops humilis (Doum) dans la région de Tlemcen: Inventaire et indices écologiques, 4ème Journée d’Entomologie, I.N.A, El-Harrach, Alger, Avril 17, 2000.

[7] A. Damerdji, B. Bouhellou, Entomofaune de la garrigue à Chamaerops humilis L. (Doum) (Palmacée) dans la région de Tlemcen (Algérie): Inventaire et aperçu bio-écologique, Bulletin du Muséum d’Histoire Naturelle de Marseille 60 (2002a) 37-43.

[8] A. Damerdji, B. Bouhellou, Faune des Invertébrés du Doum (Chamaerops humilis L.): Inventaire–Indices écologiques dans la région de Tlemcen (Algérie), Comm. orale. Deuxième colloque international des chaires UNESCO, Gas Natural sur le développement durable du Maghreb: Diversités biologiques, écologiques, culturelles et environnementales, Laghouat, Avril 28-30, 2002b.

[9] P.P. Grasse, R.A. Poisson, O. Tuzet, Zoologie I. Invertébrés, 2nd ed., Masson et Cie Éditeurs, 1970, pp. 630-631.

[10] J. Ledoux, A. Canard, Initiation à l’étude systématique des Araignées, Imp édit Domazan (Gard), Couverture: mâle d’Eresus albopictus Simon, Eresidae d’Afrique du Nord., 1981, pp.24-30.

[11] M. Chinery, Insectes d’Europe en Couleurs, Ed Bordas, 1983, p. 380.

[12] J.C. Pihan, Je reconnais les insects, Coll. Agir et

connaître, 1977, pp. 127, 156.

[13] V.J. Stanek, Encyclopédie illustrée des insects, Trad.

Française by Gründ, Paris, 1973, p. 548.

[14] E. Vallardi, Encyclopédie du monde animal, T.III, 1962,

pp. 159-463.

[15] J. Zahradnik, Guide des insectes. Ed. Hatier, 1984, p. 318.

[16] H. Heinzel, R. Fitter, J. Parslow, Oiseaux d’Europe, d’Afrique du Nord et du Moyen Orient, Edition Delachant et Niestlé, 1985, pp. 13-311.

[17] E. Zimmer, Guide de la faune, traduction et adaptation: Denis – Armand, N° ed: 0358. Ed. Arthaud, 1989, pp. 218-282.

[18] J. Dorst, Ecologie générale, Description de milieu et analyse factorielle des correspondances multiples, C.R. Acad. Sc. Paris 3 (11) (1984) 309-314.

[19] A. Damerdji, M. Adjlani, Contribution à l’étude bio-écologique de la formation à Ampelodesma mauritanicum Poiret, Durd et Schinz, 1895 (Diss) dans la région de Tlemcen (Algérie), Bulletin du Muséum d’Histoire Naturelle de Marseille 60 (2002) 53-60.

[20] A. Damerdji, L. Ladjmi, Contribution à l’étude biocénotique du Romarin dans la région de Tlemcen (Algerie), Premières Journées sur la Protection de l’Environnement, Université Aboubekr Belkaïd, Tlemcen, Mai 28-29, 2003.

[21] A. Damerdji, A. Djedid, Biocénose de la faune du genêt (Calycotome spinosa) dans la région de Tlemcen, IIème Journée de Protection des Végétaux, I.N.A., El- Harrach, Alger, Mars 15, 2004.

[22] A. Damerdji, D. Cheikh-Miloud, Faune de l’extrême ouest du littoral algérien: biodiversité et approche bioécologique, Forum Scientifique de S.N.V. «Environnement, santéet nutrition», Faculté des Sciences, Département de Biologie, April 17-18, 2007.

[23] A. Damerdji, S. Bechlaghem, Faune de la zone méridionale de Tlemcen: Biodiversité et approche bioécologique, Actes Séminaire International sur la Biodiversité Faunistique en Zones Arides et Semi-arides, Université Kasdi Merbah, Ouargla, 2009, pp. 200-206.

[24] A. Damerdji, K. Hadjouti, OPComposition et structure de la faune des Invertébrés dans trois stations de Cistus salvifolius L. (Cistacées) dans la région de Tlemcen, Séminaire International de Biologie Animale. (SIBA), Université Mentouri, Constantine, May 9-11, 2011.

[25] M.A. Khelil, Contribution à l’inventaire des Arthropodes de la biocénose de l’Alfa (Stipa tenacissima L., Graminées) dans la région de Tlemcen (Algérie), La Défense des Végétaux (257) (1989) 19-24.


The Floristic Diversity of the Tlemcen Southern Slope

Scrublands (Western Algeria)

Belhacini Fatima and Bouazza Mohammed

Laboratory of Ecology and Management of the Natural Ecosystems, Department of Ecology and Environment University Abou Bakr

Belkaid , Tlemcen 13000, Algeria


Abstract: This work was completed on the study of the southern slope scrublands (matorrals) of Tlemcen region in the northwest of Algeria (Western Algeria). These scrublands are under various states of degradation, made up of Quercus ilex, Juniperus oxycedrus, Thymus ciliatus, Rosmarinus officinalis. The problems sought in this study are to give the actual position of vegetable cover and in particular that of the formations to matorrals currently existing in the southern slope of the area of Tlemcen, while being based on the phytoecological aspect of the vegetable groupings which constitute this inheritance. Sampling is the first phase of work based on the analysis of the spatial variations of the structure and composition floristic and considering the nature of the problem to be treated, the authors considered to be useful to use the method Zuricho Montpeliéraine developed at the point by Braun-Blanquet, the method usually used consists to collect all the plant species and to make the list of the species on a small square of surface 100 m2 (have it minimal). The floristic readings (150 on the whole) were taken according to the method of Braun-Blanquet. Our results show that there exists a therophytisation marked by a general invasion of annual species as show us as the synergistic action of the aridity and the anthropic action generated important modifications on the level of the vegetation in the dynamic direction. This advanced degradation leads a steppisation which results in a substitution of the elements of the matorrals by species more adapted much the xericity. Key words: Scrublands, floristic, diversity, phanerophyte, chamaephyte, anthropical, Tlemcen, Algeria.

1. Introduction

The forest landscape and pre-forest in western

Algeria is undergoing fast regressive transformations

related to the various processes of degradation. This

subject [1] mentioned that it was infinitely probable

that these ecosystems regressive evolution (forests,

pre-forest and scrublands) was committed and could

become irreversible.

In the area of Tlemcen, the forest inheritance, like

that of the other Mediterranean zones, knew since

decades a continual regression due to a combined

action of the man (deforestation, overgrazing) and

climate (estival dryness, irregularity of the rains,

downpours violent). Such an evolution caused the

Corresponding author: Belhacini Fatima, Ph.D. candidate,

research field: plant ecology. E-mail: [email protected].

substitution of a mesophytic vegetation of origin, by a

xerophytic vegetation with the most various degrees.

Among the most recent work completed on the

vegetation and the anthropozoic influence in Oranie

and the area of Tlemcen, let us quote those of Alcaraz

[2], Benabdelli [3], Benabadji [4], Bouazza [5],

Aïnad-Tabet [6], Bouazza and Benabadji [7-9] and

Bestaoui et al. [10].

The biodiversity for a landscape is thus the resultant

of disturbance processes, succession and the space

organization of the environmental gradients from

which they result [11].

This study is aimed at the knowledge and inventory

of the southern slope scrublands flora of Tlemcen area

(Western Algeria). Its objective is the flora ecological

study, the biogeographic and biological significance of

these scrublands taxa.

The Floristic Diversity of the Tlemcen Southern Slope Scrublands (Western Algeria)

1168

The vegetation is thus used like the faithful station

conditions feedback; it is its global expression [12].

Although the vegetation is presented in the form of

scrublands in various states of degradation in the

southern slope of the area of Tlemcen (Zone of study),

this formation is used as field of practice works for

systematic botany and ecology, given its natural

character and its situation in ananthropized medium.

For all the species, the morphological, the biological

and the phytogeographical distributions types were

taken into account in the comprehensive analysis.


The issue sought in this study is to give the

vegetable cover actual condition and in particular that

of the scrublands formations currently existing in the

southern slope of the area of Tlemcen, taking into

account the phytoecological aspect of the vegetable

groupings which constitute this inheritance.

The choice of the stations was almost imposed to us,

it nevertheless is directed by the presence of the

scrublands formations which are our study topic.

Therefore we could choose three representative

stations in the study area.

This latter is located at the extreme west of Algeria

(Fig.1); with an altitude of 850 m and an area of

9,071.69 km2. It is limited by:

The Mediterranean in the north;

In the northeast, the wilaya of Aïn Temouchent;

In the east, the wilaya of Sidi Bel-Abbès;

In the west, Morocco;

In the south by the wilaya of Naama.

Its environment is located at the south of the wilaya

of Tlemcen. It is crossed by the road 22 connecting

north to south state.

2.1 Station 1: (Sidi-Djilali)

Located 3 km from Sidi-Djilali, right-hand side of

the wilaya country road 107 connecting Sebdou to

Sidi-Djilali, it is at 1,445 m of approximate altitude,

with a 5%-10% slope and a vegetal overrate between

35% and 40%.

The shrubby layer is represented by Thymus ciliatus,

Ulex boivinii and Rosmarinus officinalis dominance.

The herbaceous state is dominated by the following

species: Plantago lagopus, Asphodelus microcarpus,

Fig. 1 Studied area location map.


1169

Reseda alba and Paronychia argentea.

2.2 Station 2: (Boughdou)

Located at 21 km of Sebdou; before the village of

Aïn-Sfa, this station is at 1,408 m in altitude, with a

0-5% slope. This station is a scrubland where the rate

of vegetal cover varies between 30% and 45%.

On the floristic level, we notice Juniperus oxycedrus

and Quercus ilex, a shrubby layer including Ulex

boivinii and Rosmarinus officinalis and a diversified

herbaceous layer which dominates the station.

2.3 Station 3: (El Gor)

It is located at the southeast of the wilaya, it is in

the Sebdou Daïra vicinity. It is at 1,332 m altitude,

with a slope of 15%-20%. The rate of vegetal cover is

from 70%-75%, There is also a bedrock outcrop.

The shrubby layer is more or less represented with a

predominance of Cistus salvifolius. And in the raised

layer, although it is degraded by the antropozoological

action, Juniperus oxycedrus and Quercus ilex are

present.

The study zone is characterized on the climate map

from series of meteorological data provided by the

different stations: Saf-Saf, Ras El-Ma, Aïn-Safra and

El Aricha.

The data of 1913 to 1938 were obtained from

Seltzer’ sweater collection 13, those from 1984 to

2009, are provided by the weather stations located in

the region.

The Emberger 14 index Q2 is given by the Eq. (1):

(1)

where: P = average annual rainfall; M = average of

maximum of the hottest month (T + 273 K) and m is

the average of the minima of the coldest month (T +

273 K).

Fig. 2 Pluviothermal emberger climagram.


1170

The pluviothermal climagram shows a difference

between the stations located in the zone, they are

marked by more aridity and a rigorous winter. That

is:

Aïn Sefra station has changed from arid cold

winter to arid to cool winter;

El Aricha station moves from the semi arid cold

winter to arid cool winter;

Ras El-Ma station underwent a bioclimatic

stage shift from semi-arid cool winter to arid

moderate winter;

Saf-Saf station changes from the sub humid

moderated winter to semi-arid cool winter.


3.1 Systematic Composition

3.1.1 Families, Genera and Species

In the study zone, the inventory carried out made

it possible to enter 118 species belonging to 96

genera and 33 families. The represented genera are

variable; the distribution of the families is

heterogeneous.

Asteraceae, Lamiaceae and Poaceae dominate the

three stations (Sidi Djilali, Boughdou and El-Gor),

these families represent more than 36% of the

studied flora.

The other families are of small to very small

percentage and are generally mono generic and

sometimes even mono specific. So that in arid region

and Sahara, the majority of the families is

represented only by one or two genera, and the

majority of the genera is represented only by one or

two species.

3.1.2 Biological Characterization

The forms of the plants life are a privileged tool

for the description of the vegetation physiognomy

and structure. They are considered as an expression

of the flora and vegetation adaptation strategy to the

medium conditions.

The scrublands of the Tlemcen area southern

slope (western Algeria) are marked by a high

therophytesratio 47.46% and are most dominant in

Sidi Djilali (48.83%), Boughdou and El-Gor(47.95%)

Fig. 3 Composition in families, genera and species.


1171

Fig. 4 Percentage of the biological types.

Fig. 5 Percentage of the morphological types.

stations Then, we have the chamaephytes, with

28.81%, which are generally more frequent in the

scrublands and more especially, in the high altitude

scrublands especially on limestone (edaphic

xericity) and the xeric scrublands in southernmost

situation.

The geophytes are slightly represented with only

7.63%. They are represented by: Muscari comosum,

Iris tingitana, Urgine amaritima, Gladiolus

byzantinus, Stipa tenacissima, Asphodelus

microcarpus, Anagallis arvensis, Stipa parviflora

and Ornithogalum umbellatum.

Finally, the phanerophytes are less represented:

4.24% in the whole (except at El-Gor station with

5.48%). They represents the medium state changes

under the ecological and especially

anthropozoological factors actions.

3.1.3 Morphological Types

Our research revealed the predominance of the

herbaceous species (57.63%) on the woody species.

This is justified by the fact that this vegetation is

much subjected to the human pressure.

The population exploits the wood for heating

contributing thus to deforestation and to danger

setting of some species weakened by the ecological

stress.

On the hand, one notes that the annual plant

dominate the perennial.

3.1.4 Biogeographic Types

The biogeographic spectrum, set according to the

total floristic list of the territory, highlights the

various elements.

Among the species present at the area of Tlemcen

southern side, several have a zone of Mediterranean

distribution sphere. To study the species distribution

we used information provided by the “New Flora of

Algeria” [15].

From the chorologic point of view, the percentage

taxa with Mediterranean distribution is high enough,

namely 34.75% of the total roll. This result is in

agreement with that obtained on the whole of the

flora of the area of Tlemcen by other researchers

(Research laboratory on ecology and management of

the natural ecosystems).

Eurasia, endemic north-African and

paleotemperated taxa, occupy an appreciable rank in

the study zone, they constitute 5.93%, 4.24% and

4.24% of the global rolls, respectively.

Station of Sidi-Djilali Station of Boughdou Station of El-Got

Station of Sidi-Djilali Station of Boughdou Station of El-Got


1172

Fig. 6 Distribution of the biogeographic types.

4. Conclusion

The richness of the Tlemcen area southern slope

scrublands in western Algeria returns to Asteraceae,

with Poaceae, with Lamiaceae and with Fabaceae

recognized by their resistance to the rigour of the

climatic conditions.

The biogeographic distribution shows the

predominance of the element Mediterranean

(34.75%) then those of west-Med with 8.47%.

For all the types of raised formations and

chamaephytic, the therophyte have the highest rate,

which shows a strong anthropoid action.

The species distribution, expressed by adaptive

strategies vis-a-vis environmental constraints,

emphasizes that the chamaephytes and the

therophytes tend to invade the vegetal cover [9].

The general outline of the biological type, in the

stations, is: Th > Ch > He > Ge > Ph. The

phanerophytes occupy the last position, considering

their weak covering. The chamaephytes are frequent

in the Tlemcen area southern slope scrublands; their

number remains however less important than that of

the therophytes.

These last represent approximately twice that of

the chamaephytes.

This therophytisation, marked by a general

invasion of annual, is favoured by a short biological

cycle favourable to an intense vegetative activity

(3-6 months in general).

The risks of an aggravation of the impoverishment

of the floristic inheritance of our area (Tlemcen

Mounts and south of Tlemcen steppe zones) are real

[8].

References

[1] P. Quezel, G. Bonin, Leafy forests of the Mediterranean

circumference, constitution, ecology, current location,

prospects, Rev. For. Frenchwoman 3 (33) (1980)

253-268.

[2] C.L. Alcaraz, Vegetation of the Algerian west, Ph.D.

Thesis, State Univ. Perpignan, Perpignan, Paris, 1982,

p. 415 and appendices.

[3] K. Benabdelli, Development of a methodology of

appreciation of the pressure anthropozoogene on the

vegetation in the main forest of Télagh

(Algeria), Ph.D. Thesis, Aix-Marseilles III, 1983, p.

183.

[4] N.R. Benabadji, Phytoecological study of the steppes with Artemisia bleached on grass-alba ESA, and in


1173

Salsola vermiculata L. in the southwest of Sebdou (Oranie, Algeria), 1995.

[5] M. Bouazza, Phytoecological study of the steppes with Stipa tenacissima L. and Lygeum spartum L. in the south of Sebdou (Oranie, Algeria), Ph.D. Thesis, Be-Sci. Univ., Tlemcen, 1995, p. 153.

[6] M. Aïnad-Tabet, Analysis éco-floristic of the great structures of vegetation in the mounts of Tlemcen, M.Sc. Thesis, Univ. Abou-Bakr Belkaïd, Tlemcen, 1996.

[7] M. Bouazza, N.R. Benabadji, Floristic composition and anthropozoïque pressure in the south-west of Tlemcen, Rev. Sci. Techn. Constantine (10) (1998) 93-97.

[8] N.R. Benabadji, M. Bouazza, Impact of the man on the

forest in the area of Tlemcen, Méd. 22 (3) (2001)

269-274.

[9] N.R. Benabadji, M. Bouazza, Contribution to the study

of the floristic procession of the steppe in the south of

El Aricha (Oranie, Algeria), Sci. Tech. Special No. D

(2002) 11-19.

[10] K. Mesli-Bestaoui, M. Bouazza, D. Godroon, Study of the vegetable groupings of the mounts of Tlemcen and their facies of degradation by two approaches: Ecological profiles and interspecific connections (Oranie-Algeria), Sciences and Technology C (25) (2007) 71-78.

[11] B.F. Roise, Ecology of the landscape: Concept methods and applications tec., ED Doc., 1999.

[12] C. Beguin, J.M. Gehu, O. Hegg, Symphytosociology: A new approach of the vegetable landscapes, Lille. Doc. Phytos. N.S. [online] (4) (1979) 49-68, http://www.tele- botanica.org/.

[13] P. Seltzer, Climate of Algeria, Inst. Weather, and Phys. of the Earth, Univ. Algiers, 1946, p. 219.

[14] L. Emberger, A biogeographic classification of the climates, Rev. Wk. Laboratory, Club-footed, Zool. Fac., Sci., Montpellier, France, 1955, pp. 1-43.

[15] P. Quezel, S. Santa, New Flora of Algeria and the Southernmost Desert Areas, C.N.R.S., Paris, 1962-1963, p. 1170.


Tools for Protein Structure Prediction at the

bri-shur.com Web Portal

Sergey Feranchuk1, Ulyana Potapova2, Vladimir Potapov2, Dmitry Mukha3, Vladimir Nikolaev4 and Sergei

Belikov2

1. Met. Ltd., Minsk, Kiseliova 20, Belarus

2. Limnological Instiute SB RAS, Irkutsk 664033, Russia

3. Institute of Bioorganic Chemistry NAS Belarus, Minsk 220141, Belarus

4. Belozersky Institute of Physico-Chemical Biology, Moscow 119992, Russia

Received: April 03, 2012 / Accepted: May 31, 2012 / Published: October 30, 2012.

Abstract: Internet services on bioinformatics still remain a popular tool for the researchers. Here the authors present a recently developed web-site http://bri-shur.com where several tools and pipelines for protein structure prediction are implemented. The prediction of a structure for a particular protein often requires a sensitive and iterative approach, and the web-site provides an environment for this kind of work. Software that is used in the services includes both free programs available in the Internet and newly developed algorithms. The service on homology screening in PDB for a structure template is implemented using an approach that is alternative to well-known BLAST algorithm and it has some advantages over BLAST. The service on homology modeling uses well-known Nest program. The service on protein energy estimate allows selecting a best template in the set of homologs and adds a functionality of fold recognition to the environment. The design of the site simplifies several of the most useful bioinformatics routines, thus making them available to a large community of researchers. Services are provided free of charge without registration, and the user’s privacy is taken care of. Key words: Web-service, SOAP, homology modeling, homology screening, protein folding.

1. Introduction

The volume of biological data is increasing

drastically, so the question arises: how to unite all the

complex and diverse sets of bioinformatics algorithms

in a convenient manner. This question is not yet

completely answered, and there are several competing

concepts. But the most popular way to try to solve the

problem is to use web-portals that integrate several

different services.

The concepts of software-as-a-service and

distributed computing have now become even more

popular than when the first bioinformatics web-portals

were developed. And we believe that the concept still

Corresponding author: Sergey Feranchuk, Ph.D., research

fields: protein structure prediction, bioinformatics and computational linguistics. E-mail: [email protected].

has potential for further development and that new

technologies can make its realization even simpler.

New frameworks for implementing web-based user

interfaces are available, and there are libraries like

Bioruby [1] which manage arrays of biological data.

Such technologies are similar to the REST and SOAP

concepts for bioinformatics servers (for example, Ref.

[2]). So it seems worthwhile to try to develop a single

convenient web-site which can integrate some of the

available software and servers.

Development of the GeneBee algorithm was started

in the 90’s when one of the authors participated in the

development of a web server in Moscow [3]. Since

then the algorithm has been significantly improved

and adapted for sequence comparison.

In contrast to web-portals, like Mobyle [4], the

Tools for Protein Structure Prediction at the bri-shur.com Web Portal

1175

ideology of the bri-shur web-site is to provide not only

commonly accepted software, like the Blast [5]

algorithm for homology screening or the Clustal [6]

algorithm for multiple alignment, but also to

concentrate on the presentation and development of

new original algorithms. A second goal is to integrate

them into pipelines using well established external

programs.

In this paper, a pipeline for protein structure

prediction is described. Its ability to predict the

structure of proteins on the CASP8 target is

demonstrated to be competitive; however the main

aim of this pipeline is not to achieve a competitive

score among CASP participants but to provide an

advanced and convenient tool for biologists in their

routine work on structural biology.


The web-interface of the site is implemented using

the Ruby On Rails framework and the BioRuby

library. The servers where the data processing is

implemented are physically separate from the

web-interface server, and communication is achieved

using php and shell scripts by REST ideology.

The pipeline for protein structure prediction

typically consists of the following steps: (1) screening

by homology to a given primary sequence in PDB; (2)

manual refinement of the alignment; (3) construction

of a 3D model; and (4) estimation of the quality of the

model in order to select between alternative models.

The Genebee multiple alignment and screening

algorithms are used on the site [7, 8]. Input for the

algorithm is an amino acid or nucleotide sequence.

Databases of nucleotide and amino acids sequences

are installed on the server. The problem is to find the

sequences in the databases that are most similar to a

given query sequence. This problem is reduced to

another problem: to determine a value of similarity

between two sequences, one being the query sequence

and the other being a sequence from the database.

To determine a value of similarity, the two

sequences must be aligned. The alignment is built up

from local fragments of similarity without gaps

(motifs), and the following approach is used to

estimate similarity of motif:

The starting point is a matrix of similarity between

residues. The assumption is to consider each matrix of

similarity between residues as a normally distributed

random value. Then for each motif two hypotheses are

considered. The first hypothesis is that the

combination of scores between residues by the matrix

is a random value - and that the sum of the normalized

weights of these scores can be described by the rules

for a Bernoulli sequence of normally distributed

random values. The second hypothesis is that the

motif has some biological sense and that the

coincidence of the letters is not random. The random

value distribution for the first hypothesis can be

estimated numerically as a sum of the normalized

matrix values divided by the square root of motif

length. This random value should be distributed

normally. To estimate the probability of the alternative

second hypothesis, a Z-score for the first hypothesis is

used, i.e. the deviation of the sum of weights from the

mean value divided by the expected mean deviation

for this sum. Bri-shur internal terminology calls this

value “power” and it characterizes the quality of a

motif. The value of power could be calculated by

Eq. (1).

L

EwP ij

)(

(1)

where wij is a substitution score between two residues,

E is an average substitution score, σ is a mean

deviation and L is a length of motif.

A special algorithm, called “DotHelix” [7], is used

to select all “strong” motifs between two sequences.

After these motifs are selected, there are two methods

for making the resulting alignment, where gaps can be

inserted between motifs.

The first is to connect the motifs into clusters. This

method selects the best fragment of similarity between

sequences.


1176

The second method is to use dynamic programming

to align sequences as a whole, this is the ideology

used by Needleman-Wunsch et al. [9], with the

modification that the resulting alignment can be based

only on the previously selected motifs. PDB screening

uses the second method to find a global alignment.

To improve the quality, the algorithm of screening

was modified in comparison with Ref. [8], and PDB

screening uses a second iteration with a

position-specific scoring. The first iteration is

performed on all non-redundant subsets of Genbank

and the alignment is built up relative to the query

sequence. The score for a position is given by Eq. (2).

mSSlpk

kl

k

k

)/log()( (2)

where m is the average frequency for a given letter, S

is the distance between the query sequence and a

particular sequence with a given substitution. The

score is then normalized to be similar to a normal

distribution with mean = 0 and dispersion = 1, to be

used in the conventional motifs search procedure. The

first iteration can be performed either by the Genebee

algorithm, or by 3 iterations of a conventional

PSI-BLAST [5].

The search for the optimal respective positions

between the query sequence and the sequence from

the database is a major bottleneck in the process of

homology screening. So, to optimize execution time,

the use of GPU acceleration in this part of the program

is being developed.

The homology screening in PDB almost always

finds one or two good templates. However, when

there are no close homologs for a query sequence,

there can be a choice between a larger number of less

good templates. The choice might even be between

several fold classes, in which case, one needs to use

additional reasoning in order to choose the best fold

type for the query sequence. Therefore Bri-shur has

implemented a service for estimating the folding

energy of a given structure.

Hydrophobic energy and the energy of electrostatic

interactions make major contributions to the free

energy of a protein structure. These terms closely

balance the loss of conformational entropy of a folded

structure. A good estimate for the conformational

entropy cannot be made in a reasonable time, so the

bri-shur service gives only the relative values for

electrostatic energy and hydrophobic energy. However,

to estimate the entropic term, an average value is

subtracted from each energy to make the differences

between “good” and “bad” folds more clear. It follows

from Baldwin [10], that an estimate of the free energy

can be satisfactory only if the electrostatic energy term

and the hydrophobicity term have the same order of

magnitude. If highly polarizable force fields are used

(e.g. Amber 99) the energy of formation of the

secondary structure is implicitly included in the

electrostatic energy. To estimate the hydrophobic term in the energy

function, the solvent accessible surface area (SASA) is

first calculated for each atom. Because polar atoms are

less hydrophobic than neutral atoms in the SASA sum

for the residue, the areas of polar atoms are weighted

0.5 and the area of neutral atoms are weighted 1 [11].

A zero level value needs to be subtracted from the

SASA of the residue to get the accessible area of the

unfolded structure. This zero level value for each

residue has been pre-calculated. A subset of the

tertiary protein structures was analyzed. An

incomplete fragment of each chain was taken and the

SASA was calculated for the residues on the

assumption that this fragment was isolated; this gave

an estimate for the area of the residues in the unfolded

chain; this area depending on the length of the

fragment. The distribution of SASA for each type of

residue was used to calculate the required zero level

value.

The coulomb energy of the electrostatic interactions

in a protein gives values that are much higher than the

real folding energy, even when a value for charge

screening is subtracted. Therefore the term in the

energy function which arises from the loss of

conformational freedom and which compensates for

these high energy values, needs to be pre-calculated.


1177

This has been done as follows: It is assumed that

the loss of conformational freedom has a constant

value that depends only on the residue type. So, for

any stable protein structure, the sum of these values,

which depend only on the amino-acid sequence, must

be less than the electrostatic energy. Thus, an

inequality equation can be written for any protein

structure, and it is a linear programming problem to

obtain values for the loss of conformational freedom

of each residue from the set of inequalities. The

objective function in the linear programming problem

is a weighted sum of these values. To solve the

problem 200 structures were taken from the PDB and

their conformational freedom values were calculated.

The resulting distribution of values is used in the

energy function.

The units of the energy estimate in bri-shur’s

service are fairly arbitrary. The SASA is measured in

units of area, and the electrostatic energy units are

multiplied by a term to give an adequate gain to the

energy. Therefore, the units are normalized because a

good model requires that the estimate should be close

to the experimental folding energy of the protein,

measured in kcal/mol, and that the contribution of

both terms should be of the same order of magnitude.

The function for energy estimate could be

expressed by Eq. (3).

);(; 1 iamberehe eECEEEE

)}({2 oiih sSCE (3)

where Ee is the energy of electrostatic interactions,

Eamber is the energy in Amber force field, ei is the

estimates for a loss of conformational freedom for

each residue, Si is a weighted SASA for each residue,

s0i is the average SASA for a given residue type, C1

and C2 are scaling coefficients.


To improve the usability of the interface, additional

features have been added to the site:

The homology modeling itself can be done by

using Jason Xiang’s nest program from the Jackal

package [12];

In order to make models ready for molecular

dynamics simulations, a service for the prediction of

histidine protonation sites has been added; it uses the

open-source package PDB2PQR [13];

Services for the prediction of solvent

accessibility and secondary structure have been added

from the “Scratch” suite of programs;

To select the best model, multiple alignment is a

good tool; so several multiple alignment algorithms

are implemented on the site, including the original

Genebee, as well as Clustal [6], Mafft [14],

Muscle [15].

All these services can be connected with pipelines

(Fig. 1). There are also some tools for visualizing an

alignment more easily.

The performance of the homology search algorithm

on CASP8 models is summarized in Table 2. It should

be noted that the database used by bri-shur was even

older than the database at the time of CASP8.

The targets were taken from a subset of “easy”

Fig. 1 A scheme of services and pipelines in the bri-shur site.


1178

Table 1 The Performance of PDB screening on CASP8 targets.

Target Number of templates found (from 20 templates in the result list)

Number of two best templates in the two best results

RMSD of best template

Number of templates found by BLAST

T0388 12 2 0.93 13

T0389 3 2 1.96 1

T0390 5 0 1.36 4

T0391_1 0 0 1.79 0

T0392 1 1 1.56 1

T0393_2 0 0 1.97 0

T0394 0 0 1.67 0

T0397_2 0 0 1.99 0

T0398_1 9 2 0.67 8

T0400 1 1 1.32 1

T0401 0 0 2.18 0

T0402 1 0 1.62 0

T0404 3 2 0.98 0

T0406 0 0 1.96 0

T0407_1 0 0 2.27 6

T0408 1 0 2.12 0

T0411 1 0 1.85 2

Table 2 Results of folding energy estimates for a set of proteins with known experimental enthalpy of folding.

Protein Experiment1 Energy2 Energy3 Energy4 Decoy energy5 Decoy energy6 Decoy energy7

BPTI -1.35 -0.86 -0.36 -1.22 -0.47 -0.23 -0.70

Barnase -1.59 -1.23 -0.42 -1.65 -0.38 -0.22 -0.60

Myoglobin -1.66 -1.09 -1.38 -2.47 0.33 -0.31 0.02

Lysozyme -1.73 -1.28 -0.32 -1.60 -0.33 -0.12 -0.45

Cytochrome c -1.50 -1.23 -0.53 -1.76 -0.90 -0.33 -1.23

Ubiquitin -1.66 -1.00 -0.32 -1.32 -0.90 -0.36 -1.26 1: experimental enthalpy of folding, according to Ref. [17], in kcal/mol; 2, 3, 4: hydrophobic energy, electrostatic energy and total energy estimate for a correct model; 5, 6, 7: hydrophobic energy, electrostatic energy and total energy estimate for a decoy.

targets, by Y. Zhang’s classification [16].

Table 1 was filled as follows: the primary

sequences of CASP targets were used for screening by

both bri-shur and blast methods to find homologies in

PDB. The list of results were compared with the list of

reference templates from CASP web site for each

target. The numbers of matches were summarized in

the table, together with root mean square deviation

(RMSD) from best template in CASP to the target

model.

Among the above results, some are negative; these

poor cases might be due to the incompleteness of the

database used, which was even older and less

complete than the CASP8 database, or it might be due

to a combination of database incompleteness and

algorithm performance.

In summary, the algorithm shows an ability to find

correct templates with RMSD < 1.5 and its

performance is comparable to a conventional BLAST

search.

The results of predictions of folding energy

accomplished by means of the bri-shur web-service

are summarized in the Table 2, where the energy

values for a correct fold and for a decoy are compared.

The proteins were selected following [17], where

experimental enthalpies of folding were listed for a set

of proteins. The decoys chosen are those which, in the

homology screening, were the closest matches to a

correct fold. The units of energy are somewhat

arbitrary, because the estimate is too approximate to

be declared a “correct” energy. However the

normalizing factors are chosen so that an estimate for


1179

a correct structure has a value similar to that of an

experimental folding energy.

4. Conclusions

Summarizing the results, it can be concluded that

the bri-shur site is useful and convenient for making

pipelines for protein structure prediction. It comprises

several advanced original algorithms as well as the

conventional tools that are widely used today.

Together they serve as component parts of pipelines

for spatial structure prediction and sophisticated

analysis of proteins. In addition, the site’s new

approach to bibliographic data mining is becoming

popular as a separate tool. The developers hope that

the other services on the site will be helpful and that

the site will contribute to the development of the

internet bioinformatics community.

Acknowledgments

The authors thank Mr. Colin H. Brown and Prof.

Alexander Tuzikov for their support during this work.

References

[1] N. Goto, P. Prins, M. Nakao, R. Bonnal, J. Aerts, T. Katayama, BioRuby: Bioinformatics software for the ruby programming language, Bioinformatics 26 (20) (2010) 2617-2619.

[2] S. Pillai, V. Silventoinen, K. Kallio, M. Senger, S. Sobhany, J. Tate, et al., SOAP-based services provided by the European Bioinformatics Institute, Nucleic Acids Research (33) (2005) W25-W28.

[3] L.I. Brodsky, V.V. Ivanov, Ya.L. Kalaidzidis, A.M. Leontovich, V.K. Nikolaev, S.I. Feranchuk, et al., GeneBee-NET: Internet-based server for analyzing biopolymers structure, Biochemistry 60 (8) (1995) 923-928.

[4] B. Néron, H. Ménager, C. Maufrais, N. Joly, J. Maupetit, S. Letort, et al., Mobyle: A new full web bioinformatics framework, Bioinformatics 25 (22) (2009) 3005-3011.

[5] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z.

Zhang, W. Miller, et al., Gapped BLAST and PSI-BLAST: A new generation of protein database search programs, Nucleic Acids Research 25 (17) (1997) 3389-3402.

[6] M.A. Larkin, G. Blackshields, N.P. Brown, R. Chenna,

P.A. McGettigan, H. McWilliam, et al., Clustal W and

Clustal X version 2, Bioinformatics 23 (21) (2007)

2947-2948.

[7] L.I. Brodsky, A.L. Drachev, A.E. Gorbalenya, A.M.

Leontovich, S.I. Feranchuk, A novel method of multiple

alignment of bio-polymers (MA-tools module of

GeneBee package), Biosystems 30 (1) (1993) 65-79.

[8] V.K. Nikolaev, A.M. Leontovich, V.A. Drachev, L.I.

Brodsky, Building multiple alignment using iterative

analyzing biopolymers structure dynamic improvement of

the initial motif alignment, Biochemistry 62 (6) (1997)

578-582.

[9] S.B. Needleman, C.D. Wunsch, A general method

applicable to the search for similarities in the amino acid

sequence of two proteins, Journal of Molecular Biology

48 (3) (1970) 443-453.

[10] R.L. Baldwin, Energetics of protein folding, Journal of Molecular Biology 371 (2) (2007) 283-301.

[11] H. Zhou, Y. Zhou, Stability Scale and atomic solvation parameters extracted from 1023 mutation experiments, Proteins 49 (4) (2002) 483-492.

[12] D. Petrey, X. Xiang, C.L. Tang, L. Xie, M. Gimpelev, T. Mitors, et al., Using multiple structure alignments, fast model building, and energetic analysis in fold recognition and homology modeling, Proteins 53 (6) (2003) 430-435

[13] D.C. Bas, D.M. Rogers, J.H. Jensen, Very fast prediction

and rationalization of pKa values for protein-ligand

complexes, Proteins 73 (3) (2008) 765-783.

[14] K. Misawa, K. Kuma, T. Miyata, MAFFT: A novel

method for rapid multiple sequence alignment based on

fast Fourier transform, Nucleic Acid Research 30 (14)

(2002) 3059-3066.

[15] R.C. Edgar, MUSCLE: Multiple sequence alignment with

high accuracy and high throughput, Nucleic Acids

Research 32 (5) (2004) 1792-1797.

[16] Y. Zhang, I-TASSER: Fully automated protein structure

prediction in CASP8, Proteins 77 (9) (2009) 100-113.

[17] P.L. Privalov, A.I. Dragan, Microcalorimetry of

biological macromolecules, Biophysical Chemistry 126

(16) (2007) 16-24.


The Elaboration of Horse Meat Products Technology

Аbzhanova Sholpan, Kizatova Мaigul, Мukhtarkhanova Rauan, Тarakbaeva Raushan and Аbilmazhinova Nazum

Faculty of Food Production, Almaty Technological University, Almaty 050012, Kazakhstan

Received: December 26, 2011 / Accepted: August 13, 2012 / Published: October 30, 2012.

Abstract: In Kazakhstan, production of meat has traditionally been considered one of the main priorities in agriculture. In this case

the main national source of traditional raw meat materials is lamb and horse meat. The solution of a food problem about providing the population of the country with high-grade food protein, expansion of the range of food, increase of their biological and food value, and also creation of products meeting the requirements of a healthy food of the population, are actual problems of modern society. One of the available ways to solve these problems is the development of technology for production of various products combined with physico-biological technologies. In this regard, particular interest is creation of the combination of meat and vegetable raw materials. The aim is to develop the technology of molded meat product with the use of plant materials. Key words: Horse meat, pumpkin, ordinary meat, kazy, zhaya, sausage, vitamins, sauce, protein.

1. Introduction

Saturation of the market of food is a priority in the

economic development of the RK, which allows

people to provide high-quality food produced

domestically [1, 2].

In recent years, the world has been widely

recognized that development of a new trend in the

food industry [3, 4], the so-called functional food,

which implies the use of such products of natural

origin when used systematically have a regulating

effect on the whole body or some of the systems and

organs.

Production of products with functional purpose is

an important task for the modern food industry. On a

global scale is constantly working to develop new

functional food products that have a wide range of

applications, and point to a specific body orientation,

biotope system disease [5].

An important role in the development of population

food owned brand new, balanced in composition of

products, enriched with functional ingredients.

In turn, the development of traditional and new

Corresponding author: Kizatova Maigul, Ph.D., Prof.,

research field: food production. E-mail: [email protected].

food products based on soy protein preparations of the

protein promotes the rational use of resources and thus

is one of the most effective ways to address the

shortage of protein in Kazakhstan [1, 2].

With a view to rational and economic use of raw

meat industry, improving nutritional value of the

products and expanding their product range seems

necessary: the production of protein and dressers

texture with a wide range of functional properties, to

carry on an industrial basis, are widely used fortifier

animal and plant origin in the production of meat

combined products, better use of secondary raw meat

and vegetable raw materials industry in the production

of protein dressers [6].

The aim of the present study was to develop

technology to produce molded meat product functionality.

The practical significance of the confirmed acts

extended tastings and working off the production

technology developed in an industrial environment.


Determination of chemical composition makes it

possible to get an idea of the quality of meat and meat

products, their nutritional value, depending on the


1181

proportion of moisture, protein, fat and minerals.

Determination of total chemical composition of a

sample was produced by the test sample.

Determination of protein content: according to State

standard 25011-81, protein content was determined by

calculation using the formula: X = 100 - (X1 + X2 + X3) (1)

where, X: protein content, %; X1: moisture

content, %; X2: the fat content, %; X3: the ash

content, %.

In carrying out experimental studies, standard

methods of research were used and protein,

organoleptic characteristics and the output of finished

products were determined. The validity of scientific

results is confirmed by 3-5 times repeated

experiments.


3.1 Rationale for the Introduction of Soy Isolate in a

Molded Meat Product

Introduction to the components of meat products,

giving them dietary, prevention, and functional

properties that will solve the problem of deficiency of

essential nutrients, and give the finished product

defined positive.

Studies were performed on samples of meat

products made from lamb and horse meat, hypodermic

brine concentration of 15% with different content of

soy isolate. Control sample was a brine containing no

protein supplement. Subsequently, samples were heat

treated under identical conditions.

In order to determine the level of rational

administration of soy isolate in molded meat products,

instrumental methods of analysis was conducted in

quality and consumer properties of products, including

nutritional values of food and molded products.

Among these are the main indicators of quality

merchandise, on which the consumer makes the initial

judgment about the quality of the product. In this

regard, the analysis was performed for organoleptic

product, the resulting estimate is presented in Table 1.

According to a tasting of the highest ratings

received prototypes of molded meat products

containing soy isolate, 0.5% and 1.5% compared with

the reference product, which is characterized by

separating of free water when cutting slices of the

product and swelling on the surface of the broth

products. For a test sample with 2.5% soy isolate-mi

detected defect is the presence on the cut surface of

individual zones with high concentrations of protein in

gel form, which affects the quality of the products.

Analysis of the data in Table 1, chemical

composition suggests that the introduction of raw

meat soy isolate in excess of 0.5% accompanied by an

increase of mass fraction of protein in the finished

product, including through the introduction of protein

has higher heat-resistant than muscle, which is

confirmed by the data to determine the polypeptide

and the residual nitrogen and modeling studies of

thermal stability of soy isolate. The consequence is an

increase in total moisture content of 2.3% and 3.8%. It

is found that in samples of items, the fat:protein ratio

Table 1 Effect of the level of introduction of soy isolate in the organoleptic evaluation and chemical composition of molded meat products.

Indicators Content of soy isolate, by weight of raw material, %

The total score 0 0.5 1.5 2.5

Mass fraction of protein, % 15.78 ± 0.3 17.01 ± 0.3 17.12 ± 0.4 17.18 ± 0.4

Mass fraction of moisture, % 66.10 ± 0.8 68.10 ± 0.7 69.10 ± 0.7 69.55 ± 0.8

Mass fraction of fat, % 13.30 ± 0.2 10.11 ± 0.1 9.10 ± 0.2 8.46 ± 0.1

Mass fraction of carbohydrates, % 3.32 ± 0.2 3.04 ± 0.3 2.96 ± 0.3 3.16 ± 0.2

Mass fraction of ash, % 1.50 ± 0.2 1.74 ± 0.2 1.72 ± 0.2 1.65 ± 0.2

The energy value, kcal/100g 194.2 177.8 169.2 153.1

Score 4 4.5 4.9 4.2


1182

was improved, thereby calorie products were reduced.

In assessing the quality of molded meat products

Table 2, revealed that the experimental samples are

characterized by higher pH, which in combination

with the presence of dissolved non-denatured protein

leads to a change in the ratio of free and bound water,

and consequently reduce losses during thermal

processing and improve the organoleptic

characteristics and increase output. It is for advanced

products containing soy isolate, 0.5%, 1.5% and 2.5%,

respectively, 86.6%, 89.2% and 92.1% compared to

81.8% in the control product, which confirms the

usefulness of soy isolate.

Thus, the findings suggest that the use of soy

isolates for molded meat products improves the

quality of the finished product, moisture-binding

capacity and yield.

Based on the research and analysis, production

testing the technology of molded meat products with

plant-protein brine.

3.2 The Technological Process

Preparation of raw materials: raw materials after a

veterinary inspection, cleaning and wet toilet is cut at

a room temperature 10-12 °C and a relative humidity

above 70%. Cutting, trimming, partitioning of meat

produced in accordance with the current technological

instruction. The trimmed meat is weighed and

subjected to the ambassador.

Ambassador of the raw material: preparation of salt

food (ST RK state sandard R 51574-2003), cooking

vegetable-protein brine. In the present experiments,

the authors used the method of salting meat in the

large-lump form a concentrated solution of salt

density of 1.10 g/cm3 with the content of boiled salt

15%. To prepare a concentrated solution of sodium

chloride per 100 kg of cold water take 35 kg of salt,

mix thoroughly, allow to stand for a solution for

settling the impurities and the density of the test with

a hydrometer. The solution was filtered before use

through a layer of gauze and cooled to a temperature

not higher than 4 °C. At 100 kg of raw material was

added 8.5 kg of concentrated salt solution (normal salt

2.2 kg of water 6.3 kg).

Extrusion: extrusion plant-protein brine (ρ = 1.100

kg/m3; 0.075% NaNO2; 2.5% sugar + 20% protein

suspension) of 15% mixing.

Mixing meat with brine produced in the mixer for

2-3 minutes and leave to the uniform distribution of

salt and total absorption of its meat. Duration

ambassador is 8-10 hours. Soy protein and milk

powder hydrated just before cooking the meat in 1:2

ratio with the buttermilk.

Shaping the meat: raw material was molded into

special molds for recipes (two different versions),

taking into account a balanced diet.

Heat treatment: heat treatment carried out by

standard methods. Cooking at t = 95-100 °C (in water),

for 1.5-2 hours and 50 minutes for 1 kg of product.

Baking at t = 160-180 °C for 2-3 hours. Pressed 2-3

hours for draining fat and broth.

Cooling: the finished molded product is cooled at t

= 2-4 °C.

Quality control of finished products: controlling

weight of finished products produced at scales for

static weighing up to 1.0 g.

Table 2 Effect of introduction of the level of RBR on the physico-chemical properties of molded meat products.

Indicators Content of soy isolate by weight of raw material, %

5 10 15 20

Mass fraction of total moisture, % 65.10 ± 0.07 66.80 ± 0.08 68.40 ± 0.08 69.80 ± 0.07

Loss on heat treatment, % 18.19 ± 0.40 6.26 ± 0.40 13.11 ± 0.50 11.31 ± 0.50

рН 5.80 ± 0.03 5.84 ± 0.02 5.88 ± 0.05 5.90 ± 0.04

The value of penetration, penetration units 57.00 ± 1.30 63.90 ± 1.20 71.50 ± 1.40 82.70 ± 1.30

Mass fraction of sodium chloride, % 2.75 ± 0.20 2.65 ± 0.10 2.52 ± 0.10 2.47 ± 0.20


1183

Packing, packaging, labeling: packaging materials

must comply with current regulatory documentation,

and ensure the preservation and presentation of the

product during transportation and storage.

In each box or container products are placed one

name. Packaging products of different denominations

together produce only in agreement with the

consumers.

Meat products are sold through the retail trade, the

consumer must be marked with the general

requirements for the content of information on ST RK

1010.

Storage at T = 2-4 °C for 78 hours and sales.

It is known that the nutritional value of food is not

only an optimal ratio of major nutrients, but also a

large extent-balance of micronutrients.

A study on the chemical and amino acid

composition of the finished molded product was

conducted which showed high biological value.

Table 3 shows that the protein content compared

with the control in the molded product, “Nazik”,

“Arai” and “Damdy” increased by 0.88%, 1.28% and

1.08%, respectively, indicating their high biological

value.

The low lipid content in new products (14.12%,

14.75%, 14.69%) compared with the control sample

(16.02%) assumes the properties of dietary products.

The imbalance of the amino acid composition of

proteins can lead to metabolic disorders, slowing its

synthesis and, consequently, slowing the growth of the

Table 3 Chemical composition of finished products.

Name of the components Meat products in the forms

ControlHorsemeat in the form of “Arai” Lamb in the form of “Nazik” Allsorts in the form of “Damdі”

Protein, % 17.52 17.12 17.32 16.24

Lipids, % 14.12 14.75 14.69 16.02

Carbohydrates, % 4.45 4.22 4.50 4.10

Water, % 62.17 62.41 61.91 62.34

Ash in % 1.74 1.50 1.58 1.30

Energy, kcal 157.03 155.46 158.54 145.94

Fig. 1 Amino acid score of meat products in the form.


1184

organism. An excess of one amino acid leads to failure

and poor digestibility of others. Insufficient supply of

polyunsaturated fatty acids of the organism causes

severe metabolic disorders, including an increase in

cholesterol level in blood plasma, reduce the intensity

of growth in children, reduced resistance to

unfavorable external and internal factors that increase

susceptibility to respiratory infections and

gastro-intestinal tract and other diseases.

Analysis of the amino acid composition of the

prototype shows that the total number of amino acids

shaped meat product in the form of mutton, “Nazik”

was 5,888 mg/100 g, in the form of assorted “Damdy”

7,328 mg/100 g, horse meat in the form of “Arai”

8,343 mg/100 g.

The predominant essential amino acids in molded

meat products are the 1,209 mg/100 g leucine, lysine

1,139 mg/100 g, the second version of 1,513 mg/100 g

leucine, lysine 1,463 mg/100 g, the third version of

1,723 mg/100 g leucine, lysine 1,739 mg/100 g.

According to the amino acid composition of molded

meat product, protein amino-acid score is designed,

which determines the ratio of the content of each

essential amino acid in the test protein to their content

in the standard of FAO/WHO (Fig. 1).

4. Conclusions

The research about the improvement of functional

meat products was carried out in Almaty

Techbological University. Formed meat product was

studied by experts LLP for “nutria-test” in the

Republic of Kazakhstan for compliance of its

chemical composition, nutritional and biological

value and safety requirements by sanitary standards

of Kazakhstan.

A standard production technology “Meat Products”

ST 39482430-01-2008 LLP was approved by

normative documents, the technology is registered in

the RSE 12.05.2008g “Kazakh Institute of Standards”

South Branch.

The developed technology of molded meat products

and vegetable-protein recipe brine recommended for

use in the food industry.

References

[1] J. Uzakov, Status of Livestock and Meat Industry in the

Republic of Kazakhstan, Meat Industry Press, Moscow,

2005, pp. 18-22.

[2] J. Uzakov, M. Iskakov, S. Apraksin, Condition of

Livestock and Meat Industry in Kazakhstan, Meat

Technology, Moscow, 2005, pp. 5-8.

[3] V. Gorbatov, The state of meat production in selected

countries and continents: A review, VNIIMP Thesis,

2002.

[4] Goats and Sheep on a Personal Yard, Rostov on Don,

Russia, 2000.

[5] N. Tikhomirov, Functional Food Technology, Moscow,

Russia, 2002, p. 216.

[6] B. Rskeldiev, M. Iskakov, Effective Technology of National Meat, Semipalatinsk, Kazakhstan, 2000, p. 187.

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