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Libyan Journal of Veterinary and Medical Sciences
(Libyan J. Vet. Med. Sci.)
ISSN 2410-9215
Editorial board
Editor-in-Chief:
Dr. Abdulhakim A. El Maghrbi
Editors: Dr. Abdelrazzag A. Elmajdoub
Dr. Mohamed H. Abushhiwa
Dr. Emad M. Bennour
(Faculty of Veterinary Medicine, University of Tripoli, Tripoli, Libya)
Scope and aims
Libyan Journal of Veterinary and Medical Sciences (Libyan J. Vet. Med. Sci.) is a
biannually journal that publishes peer-reviewed papers on the fields of veterinary and
basic medical sciences. Contributions may be in the form of original research or review
articles and case reports. The goal of this journal is to provide a platform for scientists to
promote, share and discuss various new issues and developments in all aspects of
veterinary and basic medical sciences.
i
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if necessary.
ii
ii
Libyan J. Vet. Med. Sci.
7. Discussion
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e.g. Gibson TW, Brisson BA and Sears W
(2005). Perioperative survival rates after
surgery for diaphragmatic hernia in dogs and
cats: 92 cases (1990-2002). J Am Vet Med
Assoc. 227:105-109.
Book
e.g. Ganong WF (1985). Review of medical
physiology (12th
edn). Lange Medical
Publications, Los Altos: pp 55.
Chapter in book
e.g. Thomson GR (1994). Foot-and-mouth
disease. In Coetzer JAW, Thomson GR,
Tustin RC (eds) Infectious diseases of livestock
with special reference to southern Africa Vol.
1. Oxford University Press, Cape Town: pp
825-852.
Conference proceedings
e.g. Nielsen JP and Rosendal S (1994).
Ciliostasis in the pathogenesis of atrophic
rhinitis. Proceedings of the 13th International
Pig Veterinary Society Congress, Bangkok,
Thailand, 26-30 June pp119.
Thesis
e.g. Pini A (1977). Strains of African swine
fever virus isolated from domestic pigs and
from the tick Ornithodoros moubata in South
Africa. DVSc thesis, University of Pretoria.
Online materials
Only authentic online materials should be
used.
e.g. Batey R (2003). Mycotic dermatitis
(dermatophilosis) of sheep. Online at:
http://www. austbreed.com.au (accessed 2
May 2007).
13. Case report
Case report will be considered for
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Libyan J. Vet. Med. Sci.
iii
All correspondence should be addressed to:
Dr. Abdulhakim A. El Maghrbi
Editor-in-Chief
Libyan Journal of Veterinary and Medical Sciences (Libyan J Vet Med Sci)
Faculty of Veterinary Medicine
University of Tripoli
P.O. Box 13662
Tripoli, Libya
Fax: +218 21 462 8422
E-mail: [email protected]
E-mail: [email protected]
Libyan J. Vet. Med. Sci.
iv
Contents
Laboratory Investigation on the Effect of Some Plant Extracts on the Development
of Culex quinquefasciatus. Abdulhakim A. EL Maghrbi and Mohamed M. Hosni
1-6
Molecular Analysis of Factor VIII (F8) Gene Mutations and Its Detection in Libyan
Patients with Hemophilia A. Nadia N. Essarbout, Mohamed B. Marwan, Rasem K. Alajnaf, Amel A. Essarbout, Alttaf N.
Essarbout and Refaat M. Tabagh
7-12
Study of Serotonin Transporters Gene in Sporadic Amyotrophic Lateral Sclerosis
in French Population. Julien Praline, Refaat M. Tabagh, Maïté Amy, Patrick Vourc’h, Christian Andres and Philippe
Corcia
13-17
The Impact of Wheat Rhizosphere on the Distribution of Burkholderia spp and Soil
Bacteria. Merfat T. Ben Mahmud
18-21
Clinical Outbreaks of Flavobacterium columnare among Tilapia Populations from
Lake Qarun, Egypt. Dina A. Mosselhy, Mohamed A. El-Aziz, Mona M. Husien, Abdelsalam A. Abumhara
and Alaa E.
Eissa
22-28
Effects of Mercury Chloride on Sperm Parameters and Pathological Changes in
Kidney of Mouse.
Habiba A. El Jaafari, Amr M. Fathalla and Suhera Aburawi
29-32
Libyan J. Vet. Med. Sci.
v
Bushwereb et al.
L .J. V. M. Sci. (2015) Vol. 1 (1): 1-4.
Laboratory Investigation on the Effect of Some Plant Extracts on the
Development of Culex quinquefasciatus
Abdulhakim A. EL Maghrbi¹* and Mohamed M. Hosni²
¹Department of Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Tripoli, P.O. Box 13662, Libya
²Department of Preventive Medicine, Faculty of Veterinary Medicine, University of Tripoli, P.O. Box 13662, Libya
Received 4October 2015/ Accepted 18 November 2015
Keywords: Culex quinquifasciatus, development, larvae, plant extracts
Introduction
Chemical control methods of vectors like
mosquitoes based on the application of synthetic
insecticides but due to environmental pollution and the
development of resistance by most of species of
mosquitoes including vectors of important diseases as
malaria, Bancroftian filariasis and yellow fever
(Georghiou, 1980 and Pessoa and Martins, 1988). It’s
necessary to find an alternative, cheaper, vector control
methods which require easy technology, but give
excellent results. Phytochemicals obtained from plants
with proven mosquito control potential can be used as
an alternative to synthetic insecticides or along with
other insecticides under the integrated vector control.
Plant products can be used, either as insecticides for
killing larvae or adult mosquitoes or as repellents for
protection against mosquito bites, depending on the
type of activity they possess.
A large number of plant extracts have been reported
to have mosquitocidal or repellent activity against
mosquito vectors (Sukumar et al. 1991). Biologically
active plant extracts are therefore being studied for their
potential efficacy to minimize the extent of pollution
and for its activities as larvicidal, ovicidal, influence on
oviposition behavior and on the growth of pre-imaginal
stages. Certain bioactive compounds derived from
Apium graveolens seeds have been proven to possess
nematocidal activity against Caenorhabditis elegans
and Panagrellus redivivus, antifungal activity against
Candida albican, C. kruseii and C. parapsilasis, and
mosquitocidal effects against Aedes aegypti fourth-
instar larvae (Rafikali et al. 2000; Rafikali and
Muraleednaran 2001). Choochote et al. (2004)
investigated the crude seed extract of celery, A.
graveolens, for anti-mosquito potential, including
larvicidal, adulticidal, and repellent activities against
Aedes aegypti. They found that ethanol-extracted A.
graveolens possessed larvicidal activity against fourth
instar larvae of Ae. aegypti with LD50 (81.0 mg/L) and
LD95 ( 176.8 mg/L).
For adulticidal activity, the plant extract exhibited a
slightly adulticidal potency with LD50 (6.6 mg/cm²)
and LD95 (66.4 mg/cm²). Also, this extract showed
repellency against adult females with ED50 and ED95
values of 2.03 and 28.12 mg/cm2, respectively.
Warikoo et al. (2011) revealed that the addition of
100% oil of Ocimum basilicum and Cymbopogon
nordus caused complete ovi-position deterrence of
Ae.agypti but A. graveolens caused of 75% effective
repellency. Papaya seeds, Carica papaya have
antifungal, antibacterial, anti-helminthic and anti-
amoebic properties, (Chavez 2011; Aravind et al.
2013). Masamba et al. (2003) reported that the oil of
Lemongrass, C. citratus has both contact and fumigant
toxicity effects on larger Grain Borer (Prostephanus
truncates). Also, reported that the oil Lemongrass when
applied to maize grain in storage, there were significant
reductions in maize damage and weight loss.
Sherwani et al. (2013) concluded that the crude
aqueous extract of C. citratus (lemongrass) showed
anthelmintic activity and could be apply as an effective
agent in future after further exploration. Dhanasekaran
et al. (2013) they investigated the larvicidal, ovicidal,
and repellent potential of the ethanolic crude extracts
Original article
Abstract Ethanol and acetone extracts of four species of plants (Allium tuberosum, Apium leptophylum, Carica papaya and
Cymbopogon citratus) were tested in respect of their influence on the development of pre-imaginal stages of mosquito
(Culex quinquifasciatus) in concentration 100, 10 and 1mg/L. Eight experiments, four replications were conducted in
each case. For each replication, 25 first stage larvae were placed in each dish of experiment and control. The
developments of larvae were followed daily till the completion of cycle. All concentrations of ethanol and some of
acetone extracts of A.tuberosum were significantly prolonged the time of the growth beside increase mortality of
larvae in acetone extract of concentration 100mg/L during development. A. leptophyllum in ethanol and acetone
extracts of concentrations 100mg/L, the developmental period of the pupal stage and in acetone extract of 10mg/L
from L4 to pupa were significantly lengthened. Also, the same vegetable in both extracts produce mortality during this
experiment. The two extract of C. papaya in 100mg/L and the extracts of C. citratus in 100 and 10 mg/l were
prolonged time of the development and causing high mortality, out of 100/100 and 77(100) in ethanol and acetone
extracts of C. papaya respectively but only acetone extract of C. citratus lead to high mortality of larvae 25/100 in
100mg/L. The differences in obtained responses necessitate the adoption of deeper research to isolate the active
principle of such plants for potential use in mosquito control program.
Libyan Journal of Veterinary and Medical Sciences December 2015 Vol. 1 (2): 1-6
*Corresponding Author: Abdulhakim A. El Maghrbi. Department of Microbiology and Parasitology, Faculty of Veterinary
Medicine, University of Tripoli. P. O. Box 13662 Tripoli, Libya.Tele.+218 925360665, Email: [email protected].
1
EL Maghrbi et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:1-6
. from the medicinal plants. Celosia argentea,
Anthocephalus cadamba, Gnetum ula, Solena
amplexicaulis and Spermacoce hispida against the
medically important mosquito vectors, Anopheles
stephensi, Ae. aegypti and Culex tritaeniorhynchus.
They concluded that among the five plant extract, G.
ula and S. hispida have significantly higher larvicidal,
ovicidal and repellent activity against selected human
vector mosquitoes An. stephensi, Ae. aegypti and Cx.
tritaeniorhynchus.
Coello et al. (2013) demonstrated that the
chloroform extracts of C. papaya seed has significant
antiprotozoal activity, but does not totally eliminate
Trypanosoma cruzi trypomastigotes during the active
phase of infection. Alcoholic/acetone extracts of C.
papaya and C. citratus at 100 mg/L; were proved to be
repulsive for ovi-position of Ae. fluviatilis. On the other
hand, acetone extracts of Allium tuberosum at 100 and
10 mg/L and A. leptophyllum at 100 mg/L produced
same effect on ovi-position behavior of Ae. fluviatilis.
Four acetone extracts (A. tuberosum, A. leptophylum, C.
papaya and C. Citrates) were repulsive for ovi-position
at 100 mg/L. Acetone extract of A. tuberosum at 10 and
1 mg/L and C. citratus at 10 mg/L maintained the same
properties (EL Maghrbi and Hosni 2014).
Cx. quinquifasciatus is one of the species essentially
domestic, per domestic and the principle vector on
Wuchereria bancrofti in the world (Pessoa and Martins,
1988). This study was designed to investigate the
influence of ethanol and acetone extracts of four plants
on development of pre-imaginal of Cx. quinquifasciatus
in the laboratory.
Materials and methods
Adult male (1000) and female (1000) mosquitoes
(Cx. quinquifasciatus) were maintained in laboratory in
transparent screen cages (40x40x40cm) containing 5%
honey solution and recipient contain dechlorinated
water for females oviposition at 27± 2 °C and 75 ± 5%
R.H. and 12 h photoperiod. Adult females were also fed
on pigeon. The larvae were reared in plastic containers
with approximately seven liters of clean dechlorinated
water and changed completely every two days. Larvae
were feeding on grinding, sieved and autoclaved ration
of cats.
Four species of plants, Allium tuberosum
(Liliaceae); Apium leptophylum (Umbelliferae); Carica
papaya (Caricaceae); Cymbopogon citrates (Graminae)
were selected on the basis of its biological activity on
mosquito or other organism, facility of obtaining and
plenty in nature. The plants were identified according to
Marbberly (1987). Plant extracts were prepared by
agitating the dried and ground plant parts in ethanol
and/or acetone separately for 24 h followed by filtration
and later recuperation of solvent using rotary
evaporator.
Eight experiments, four replications were conducted
in each case. For each experiment, concentration of 100,
10 and 1mg/L in distilled water of each plant extract
was used (150ml/9.5cm in diameter). To facilitating
dilution in water, every 100mg of extract were initially
dissolved in 2 ml of ethanol or acetone and then diluted
in one liter of distil water. The same solvents were
added in similar proportion in controls. For each
replication, 25 first stage larvae were placed in each
dish of experiment and control (containing distal water)
after removing the water through the filter paper to
avoid alteration the concentration of extracts.
All the test and control cups were covered with
netting to prevent successfully emerged adults from
escaping into the environment. The developments of
larvae were followed daily until the complete
emergence of adults. All changes in the different
concentrations and control were recorded. All dead
larvae and pupa were removed. The Medias which
contain the larvae were changed completely every three
days. The developed larvae were feeding on grinding,
sieved and autoclaved ration of cats with equal quantity
according to AMCA (1970).
Results and discussion
The following results of the experiments on the
development of pre-imaginal stages of Cx.
quinquifasciatus in different concentrations of ethanol
and acetone extracts of the plants were illustrated in the
figures 1 to 8 (The different letters indicating the
differences significantly occurred inside each stage
evolution) and the mortalities occurred during the same
experiments (Tables 1 to 8).
All concentrations of ethanol and some of acetone
extracts of A.tuberosum were significantly prolonged
the time of the growth beside increase mortality of
larvae in acetone extract of concentration 100mg/L
during development, figure (1-2) and Table (1), (2).
Figure 1. Effects of ethanol extract concentrations of
Allium tuberosum on the development of pre-imaginal
stages of Culex quinquefasciatus.
Figure 2. Effects of acetone extract concentrations of
Allium tuberosum on the development of pre-imaginal
stages of Culex quinquefasciatus.
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EL Maghrbi et al.
Libyan J. Vet. Med. Sci. Vol. 1 (2) December 2015:1-6
Table 1: Number of mortality, males and females of Cx. quinquefasciatus developed in
various concentrations (mg/L) of ethanol extract of Allium tuberosum and in control (0).
Mg/L Larval
mortality
Pupal
mortality
Total
mortality
Adults
emerged Male Female
100 4 3 7 93 41 52
10 3 4 7 93 45 48
1 1 2 3 97 52 45
0 1 6 7 93 46 47
In each concentration 4 replications of 24 larvae
Table 2: Number of mortality, males and females of Cx. quinquefasciatus developed in
various concentrations (mg/L) of acetone extract of Allium tuberosum and in control (0).
Mg/L Larval
mortality
Pupal
mortality
Total
mortality
Adults
emerged Male Female
100 44 - 44 56 29 27
10 2 1 3 97 43 54
1 - 1 1 99 45 54
0 1 1 2 98 51 47
In each concentration 4 replications of 24 larvae
A.leptophyllum in ethanol and acetone extracts in
concentration 100mg/L, the pupation period were
delayed and in acetone extract of 10mg/L, the time of
the growth from L4 to pupa were significantly
lengthened. Also, the same vegetable in both extracts
produce mortality during this experiment, figure (3), (4)
and Table (3), (4).
Figure 3. Effects of ethanol extract concentrations of
Apium leptophyllum on the development of pre-
imaginal stages of Culex quinquefasciatus.
Figure 4. Effects of acetone extract concentrations of
Apium leptophyllum on the development of pre-
imaginal stages of Culex quinquefasciatus.
Both extracts of C. papaya in 100mg/L, the time of
the growth were increased and causing high mortality,
out of 100/100 and 77/100 in ethanol and acetone
extracts respectively, figure (5)-(6) and Table (5)-(6).
Figure 5. Effects of ethanol extract concentrations of
Carica papaya on the development of pre-imaginal
stages of Culex quinquefasciatus.
Figure 6. Effects of acetone extract concentrations of
Carica papaya on the development of pre-imaginal
stages of Culex quinquefasciatus.
Table 3: Number of mortality, males and females of Cx. quinquefasciatus developed in
various concentrations (mg/L) of ethanol extract of Apium leptophyllum and in control (0).
Mg/L Larval
mortality
Pupal
mortality
Total
mortality
Adults
emerged Male Female
100 13 7 20 80 45 35
10 - - - 100 48 52
1 - 2 2 98 57 41
0 1 - 1 99 50 49
In each concentration 4 replications of 24 larvae
3
EL Maghrbi et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:1-6
. Table 4: Number of mortality, males and females of Cx. quinquefasciatus developed in
various concentrations (mg/L) of acetone extract of Apium leptophyllum and in control (0).
Mg/L Larval
mortality
Pupal
mortality
Total
mortality
Adults
emerged Male Female
100 69 2 71 29 16 13
10 3 2 5 95 46 49
1 6 - 6 94 52 42
0 6 - 6 94 53 41
In each concentration 4 replications of 24 larvae
Table 5: Number of mortality, males and females of Cx. quinquefasciatus developed in
various concentrations (mg/L) of ethanol extract of Carica papaya and in control (0).
Mg/L Larval
mortality
Pupal
mortality
Total
mortality
Adults
emerged Male Female
100 100 - 100 - - -
10 7 6 13 87 42 45
1 2 8 10 90 48 42
0 5 4 9 91 47 44
In each concentration 4 replications of 24 larvae
Table 6: Number of mortality, males and females of Cx. qyuinquefasciatus developed in
various concentrations (mg/L) of acetone extract of Carica papaya and in control (0).
Mg/L Larval
mortality
Pupal
mortality
Total
mortality
Adults
emerged Male Female
100 77 - 77 23 10 13
10 9 - 9 91 52 39
1 4 3 7 93 50 43
0 6 3 9 91 43 48
In each concentration 4 replications of 24 larvae
The concentrations 100 and 10mg/l of both extracts
of C.citratus were delayed the time of the development
and lead to high mortality of larvae in high
concentration of acetone extract only (Figure (7), (8)
and Table (7), (8).
Figure 8. Effects of acetone extract concentrations of
Cymbopogon citratus on the development of pre-
imaginal stages of Culex quinquefasciatus.
Figure 7. Effects of ethanol extract concentrations of
Cymbopogon citratus on the development of pre-imaginal
stages of Culex quinquefasciatus.
Table 7: Number of mortality, males and females of Cx. quinquefasciatus developed in
various concentrations (mg/L) of ethanol extract of Cymbopogon citrarus and in control (0).
Mg/L Larval
mortality
Pupal
mortality
Total
mortality
Adults
emerged Male Female
100 11 1 12 88 39 49
10 8 3 11 89 41 48
1 7 3 10 90 48 42
0 6 1 7 93 50 43
In each concentration 4 replications of 24 larvae
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EL Maghrbi et al.
Libyan J. Vet. Med. Sci. Vol. 1 (2) December 2015:1-6
Table 8: Number of mortality, males and females of Cx. quinquefasciatus developed in
various concentrations (mg/L) of acetone extract of Cymbopogon citrarus and in control (0).
Mg/L Larval
mortality
Pupal
mortality
Total
mortality
Adults
emerged Male Female
100 25 1 26 74 30 44
10 6 4 10 90 49 41
1 1 3 4 96 55 41
0 3 2 5 95 46 49
In each concentration 4 replications of 24 larvae
The influence of vegetable or its extracts on the
population of mosquitoes were aboard by number of
authors with different forms; Supavaran et al. (1974)
found that between 36 methanol extracts, 12 reduced
the emerging of the adults, 11 inhibited significantly the
development of larvae and 10 prolonged the time of
growth of the larvae of Aedes aegypti.
Also, the authors reported that the methanol extract
of Ricinus communis was capable to kill all larvae (L4)
of Ae. aegypti in three days in concentration of 1000
ppm. Osmani and Sighamony (1980) reported that the
oil of Cymbopogon when tested against Ae. Aegypti in
Bangalore in India induced high mortality for larvae
(L3 and L4) but considered poor ovicidal and no
produce any significant effect in relation to inhibition of
the growth. In our results, the ethanol and acetone
extracts of this plant were produced significant effect on
the growth of Cx.quinquefasciatus and have the same
effect on the larvae in acetone extract only. Mwangi and
Rembold (1988) reported that water extract of Melia
volkensii inhibited the growth of second stage larvae of
Ae.aegypti.
EL Maghrbi (2013) reported that the ethanol extract
of C.citratus, acetone extract of A. leptophyllum and C.
citratus at 100mg/L were capable of reducing the
number of eggs hatched significantly, in addition
ethanol extract of A. tuberosum, C. papaya and acetone
extract of A. tuberosum at 100mg/L were significantly
kills the larvae of Ae. fluviatilis. Kloss et al. (1987)
found that water extract of seeds of C.papaya have low
to moderate moluscide (Biomphalaria pfeifferi) in
Kenya. Water extract of C.papaya has toxic activity on
the larvae of Cx. quinquefasciatus (Evans and Raj,
1988). Oil of C.citratus when tested against Ae.aegypti
in India, was able to induce high mortality to larvae
(Grandi et al. 1989).
Rahuman et al. (2009) investigated the larvicidal
potential of indigenous plant extracts from commonly
used medicinal herbs as an environmentally safe
measure to control the filarial vector Cx.
quinquefasciatus, they found that the all plant extracts
showed moderate larvicidal effects after 24 h of
exposure at 1.000 ppm; however, the highest larval
mortality was found in stem-park hot water, acetone
and methanol extracts of Cedrus deodara
(LC50=133.85, 141.60 and 95.19 ppm, LC90= 583.14,
624.19 and 639.99 ppm.) and leaf hot water, acetone,
methanol and chloroform extracts of Nicotiana tabacum
(LC50= 76.27, 163.81 and 83.38 and 105.85ppm,
LC90= 334.72, 627.38, 709.51 and 524.39 ppm) against
the larvae of Cx. quinquefasciatus respectively.
Consuli et al. (1991) recorded that some ethanol
extracts of 17 species of plants at 100 mg/L produced
low percent of egg hatching laid in these extracts. Also,
11.1% and 33.3% of ethanol extracts influenced
surviving of larva of Cx.quinquefasciatus and
Ae.fluviatilis respectively and 11.1% of acetone extracts
for both species. Zebitz (1984) denoted that aqueous
extract obtained with organic solvent of Azadirachta
indica caused extreme prolongation of larval period,
when L1 exposed to these extracts, this author
suggested that the component of Azadirachtin and
others presented in this vegetable being capable the
interfere in hormonal equilibrium or affect on neuro-
endocrine control of ecdisteroides.
In Algeria, A. indica extract, was tested against
larvae and pupae of Cx. pipiens under laboratory
conditions. After treatment of larval stage, LC50 and
LC90 values for Azadirachtin were 0.35 and 1.28 mg/L
in direct effect and 0.3–0.99 mg/L in indirect effect,
respectively. Also, after treatment of the pupal stage,
the LC50 and LC90 in direct effect were measured as
0.42–1.24 mg/L and in indirect effect was 0.39–1.14
mg/L respectively. In addition, mosquito adult
fecundity was decreased and sterility was increased by
the Azadirachtin after treatment of the fourth instar and
pupal stage. The treatment also prolonged the duration
of the larval stage (Alouani et al. 2009).
It is worthy to perceive that extract concentration
and type of solvents for extraction are important when
used to influence the development. In conclusion, our
results indicated that the plants used in the current study
were rich source of valuable compounds. Therefore,
screening of these plants will be of great interest and
further investigation should be undertaken to identify
the biological active compound and their chemical
structure.
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Mosquitocidal, nematicidal and antifungal
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Entomol Exp Appl. 35:11-16.
6
Bushwereb et al.
L .J. V. M. Sci. (2015) Vol. 1 (1): 1-4.
Molecular Analysis of Factor VIII (F8) Gene Mutations and Its Detection in
Libyan Patients with Hemophilia A
Nadia N. Essarbout1 Mohamed B. Marwan
2,3,4, Rasem K. Alajnaf
5, Amel A. Essarbout
1, Alttaf N.
Essarbout4 and Refaat M. Tabagh
2*
¹Department Of Public Health / Faculty of Medical Technology, University of Tripoli, Tripoli-Libya
²Department of Zoology, Faculty of Science, University of Tripoli, Tripoli-Libya
³Department of Paediatric Oncology Hematology, Tripoli Medical Center, Tripoli-Libya
4Group of Blood Researchs, Biotechnology Research Center, Twesha, Libya 5Polyclinic of Airport Road, Tripoli, Libya
Received 8 October 2015/ Accepted 18 November2015
Key words: Factor VIII, Hemophilia A, PCR, Restriction enzyme, Exons 23, 24, and 26 A+B, Molecular analysis
Introduction
The X-linked coagulation disorder hemophilia A
(HA) is caused by mutations in the factor 8 (F8) gene,
this gene encodes coagulation factor VIII (FVIII) and is
located at Xq28, in the end of the long arm of the X
chromosome, telomeric region (Alan et al.1987).The
patients with severe hemophilia A have a factor VIII
(FVIII) activity level of less than 0.01 IU/ mL. Severe
hemophilia has changed from a debilitating disease to a
condition with a good quality of life (Dorain et al.2003;
Santacroce et al.2008). Studies show great advances in
efficacy and safety of FVIII products and treatment
strategies have made this possible, Factor VIII (FVIII)
is an essential blood-clotting protein, also known as
anti-hemophilic factor (AHF) (Plug et al.2008).The F8
gene mutation type is the most important predictors of
the risk of inhibitor development in hemophilia A
(Scwaab et al. 1995; Ter et al.2008).
Gouw et al. (2012) reported that the meta-analysis
confirm earlier reports that the F8 genotype is an
important determinant of inhibitor development in
patients with severe hemophilia A. The inhibitor risk in
large deletions and nonsense mutations was higher than
in intron 22 inversions, the risk in intron 1 inversions
and splice site mutations was equal and the risk in small
deletions/insertions and missense mutations was lower.
Coagulation factor VIII is made chiefly by liver
sinusoidal cells and endothelial cells throughout the
body (Kumaran et al. 2005). This protein circulates in
an inactive form in the bloodstream and bound to the
other molecule called von Willebrand factor, until
occurs damages in blood vessels. When injuries take
place, coagulation factor VIII is activated and separates
from von Willebrand factor (Sadler et al, 1998). The
severity of hemophilia A is determined by the level of
clotting activityof factor VIII in the blood (Millar et al.
1990).
In haemophilia A patients with the same mutation in
the F8 gene show heterogenous bleeding phenotypes.
Specific mutations in the A2 domain of factor VIII are
associated with mild haemophilia and a higher risk of
inhibitor development, so a double mutation in mild
haemophilia A are rarely reported (Bakija et al.2005).
In general Hemophilia A is a single gene disorders,
this group is determined primarily by a single mutant
gene and follows the Mendelian pattern of inheritance.
Hemophilia is caused by mutation in the F8 gene.
Hundreds of mutations in this gene have been
identified. These span a diverse range of mutation
types, namely, missense, splice-site, deletions of single
and multiple exons, inversions, etc. The gene for factor
VIII was cloned in 1984. The factor VIII gene size is
186 kb and contains 26 exons and 25 introns. The exon
length varies from 69 to 262 nucleotides except for
exon 14 which is 3106 nucleotides long and exon 26
which has 1958 nucleotides. Also there are six long
introns of more than 14 kb, such as intron 22 which is
32 kb long. (Antonarakis and Kazazian 1988;
Antonarakis 1995).
Original article
Abstract Hemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative
and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging
because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular
studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for
this disorder. The present study aimed to obtain data on sequence variation in F8, a set of functionally validated control
chromosomes of Libyan descent; we have performed for the first time molecular analysis of F8 in 43 Libyan hemophilia
patients. This study included 63 cases 43 patient, 10 Carrier, and 10 Control, Amplifying of F8 by using PCR technique,
and identification of mutations by using restriction enzyme Taq I for exon 23. Our results showed the presence of partial
deletions in two brother patients in exon 23 this result had been concluded refer to areas that failed to be amplified by
PCR. Our study may contribute to the knowledge on the risk factors of inhibitor development that allows libyan clinicians
to assess whether an individual patient is at high risk.
Libyan Journal of Veterinary and Medical Sciences December 2015 Vol. 1 (2): 7-12
*Corresponding Author: Refaat Mohamed Tabagh, Department of Zoology, Faculty of science, University of Tripoli.
Tripoli, Libya. Tele.+ 218092 404 65 67. E.mail: [email protected].
7
Essarbout et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:7-12
. Hemophilia A is also known as classic Hemophilia
(Alan and David 1987; Gad et al.1999; Lloyd and
Samuel 1985). It is the most common type of this
disease, affecting 1 from 10,000 males worldwide (Gad
et al.1999; Mannucci and Tuddenham 2001). The
severity of hemophilia A is determined by the level of
clotting activity of factor VIII in the blood. There are
levels of severity of hemophilia, mild from 5 to 30 % of
normal, moderate from1 to 5% of normal, and severe
less than 1% of normal (Taq 2007). Hemophilia usually
affects only males, because the hemophilia gene is
carried on the same chromosome that determines
whether the person is male or female, The
chromosomes that determine the person's sex are called
X and Y. Males have an X and a Y chromosome and
Females have two X chromosomes (Plug et al.2008).
Materials and methods
Blood samples collection
This study included 43 Patient (40 Male, 3 Female),
10 Carrier, and 10 Control (8 Male, 2 Female) the total
is 63 cases. Blood samples were collected from Libyan
patients (Department of Pediatric Oncology, Tripoli
Medical Center). Five ml of blood were drawn from
each patient and collected in tube containing EDTA and
kept in freezer (-20ºC).
The blood samples were transported to DNA
Laboratory, Human's Tissues Department and the
Genetic Engineering Department , Biotechnology
Research Center, Twesha – Libya, for DNA Studies
Amplifying of F8 by using Polymerase Chain
Reaction( PCR ) technique, and identification of
mutations by using restriction enzyme Taq I for exon 23
, exon 24 , and exon 26 A+B for researching for
mutation in Exon 23, 2147 , CGA to TGA (Arg to
Stop), Exon 24 , 2209, CGA to TGA (Arg to Stop), and
Exon 26 A+B, 2307, CGA to TGA (Arg to stop).
Genomic DNA Extraction and Primers
DNA was extracted from frozen blood samples by
QIAamp DNA Blood Mini Kit from Qiagen.
Oligonucleotide primers used to amplify exons of factor
VIII are listed in table (1).
Amplification of F8 gene by Polymerase Chain
Reaction (PCR) condition: The PCR was performed in
a final volume of 50L containing 100ng genomic
DNA, 125μM each dNTP, 200 nM of primers 1.5MM
MgCl2 and 1 U of Taq DNA polymerase (Invitrogen).
The 30 -40 cycle amplification program was carried out
in a thermocycler T100 Thermal cycler BIO-RAD
(Table1).The steps of PCR were as follows:
denaturation, 30 sec at 95 ºC , annealing 30 sec at 50-60
ºC, extension 30 sec at 72 ºC and final extension step 6
min at 72 ºC (Saiki et al.1988; Saiki et al.1985).
Restriction enzyme analysis
The PCR products from the amplification were
digested with Taq 1. PCR reaction mixture 10 µL (~0.1-
0.5 µg of DNA),nuclease-free water 18 µL,10X Buffer
TaqI 2 µL, TaqI 1-2 µL, Incubate under paraffin oil in a
capped vial at 65°C for 1-16 hours. Stop the digestion
reaction by adding 0.5 M EDTA, pH 8.0 (#R1021), to
achieve a 20mM final concentration. Mix thoroughly,
add an electrophoresis loading dye and load onto gel.
The digests were electrophoresed on 2%
gelelectrophoresis and the restriction enzyme pattern
was visualized after staining the gel with ethidium
bromide (Table2) (Taq1 BIORON GmbH).
Table 1: Oligonucleotide primers for PCR Analysis of Factor VIII.
Exon 5' Primer 3' Primer Size of PCR
fragment (bp)
23 5'- GAAGGAAGATATGATTGACAGA -3' 5'- AACTAGAACAGTTAGTCACC -3' 258
24 5'- ATAACTGAGGCTGAAGCATG -3' 5'- GTAGATCTGTTGCCTCTTACC -3' 242
26(A+B) 5'- GCTTTGCAGTGACCATTGTC -3' 5'- AGCTGAGGAGGGAGAGGTGA -3' 192
Table 2: Restriction Enzyme used in this study.
Name Restriction
Enzyme
Source of Restriction
Enzyme Recognition Sequence
Number of Cuts in F8
Gene
Taq I Thermus aquaticus bacterium
BIORON GmbH 5'…T ^ CGA…3'
3'…AGC ^ T…5'
87 position
Results
Analysis of three Taq I sites in exons 23, 24 and 26
(A+B) in studied patients at Two patients with a
moderate form of the disease demonstrated an abnormal
electrophoretic pattern in exon 8 and sequencing by
DNA Sequencing Capillary Electrophoresis from
Applied Biosystem and analyzed by SeqScape
Software, this software demonstrated missense
mutations at codon 2147 for arginine within a thrombin
activation site. These mutations were C to T transitions
changing a CGA codon for arginine to a TGA
termination codon, Results had been reached in this
study showed the presence of tow partial deletions in
two brother patients in exon 23 had been conclude refer
to areas that failed to be amplified by PCR (Figure 1, 2
and 3). Previous studies showed that Partial deletion in
8
Essarbout et al.
Libyan J. Vet. Med. Sci. Vol. 1 (2) December 2015:7-12
exons of FVIII gene refer to do not amplify by PCR
(Table3).conclude refer to areas that failed to be
amplified by PCR.
Table 3: Previous studies show that Partial deletion in
exons of FVIII gene refer to do not amplify by PCR .
Exon Severity References
23 Moderate J-M Lavergene et al. (1992)
24 Moderate J-M Lavergene et al. (1992)
23-24 Moderate J-M Lavergene et al. (1992)
23-26 Sever J-M Lavergene et al. (1992)
26 Sever J-M Lavergene et al. (1992)
24-25 - Vidal et al. (2002)
25 - Vidal et al. (2002)
25-26 - Vidal et al. (2002)
23 mild In this study
Figure 1: Detection of the Partial deletion [ Exon 23, codon 2147, (Arg Stop)] by Taq I digestion of the 291bp of amplified
fragment to 145bp and 146bp this case is normal, but when does not cut is abnormal case. Lane M: DNA Marker. 7, 8,9,10 No PCR
Product caused by Partial Deletion. Lanes: 1, 2,3,4,5,6,11,12,13,14 are normal for the nonsense mutation of codon 2147, (Arg
Stop). (PP=PCR Product; RE= Restriction Enzyme; N=Normal; CM=Carrier Mother; (+) With Mutation; (-) Without Mutation;
♀=Female; ♂=Male, Taq Icut innormal allele.
Figure 2: Gel electrophoresis of the amplicons of exon 24 For FVIII gene, size of PCR Productis 224 bp
and Taq I restriction digestion gives two fragments. The size of the first fragment is 62 bp and the second
is 162 bp. Lane M DNA Ladder (50-1000 bp); Lane 1P/N ♂; 1R/N-♂; 2P/H♂; 2R/H- ♂. P=PCR Product;
R= Restriction Enzyme; N=Normal Case; H=Hemophiliacs; (-) Case Without Mutation;♂=Male;
Taq Idigested innormal allele.
9
Essarbout et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:7-12
.
Figure 3: Gel electrophoresis of amplicon of exon 26 (A+B) of FVIII gene. Size of PCR Productis 264 bp
and Taq I restriction digestion gives two fragments. The size of the first fragment is 104 bp and the second is
160 bp.(The DNA sequencing by Sequencing Capillary Electrophoresis from Applied Biosystem and analyzed
by SeqScape Software, this software) Lane M DNA Ladder (50-1000 bp); Lane1P/N ♂; 1R/N-♂;2P/H♂; 2R/H- ♂;
3P/H ♂; 3R/H- ♂. P=PCR Product;R= Restriction Enzyme; N=Normal Case; H=Hemophiliacs; (-) Case Without Mutation;
♂=Male; Taq Idigested innormal allele.
The patients divided into three groups according to
severity 19 Severe, 8 Moderate and 16 Mild (Table 4).
People with hemophilia A often bleed longer than other
people, bleeds can occur internally, into joints and
muscles, or externally, from minor cuts, dental
procedures or trauma. How frequently a person bleeds
and the severity of those bleeds depends on how much
FVIII is in the plasma, the straw-colored fluid portion
of blood.
Normal plasma levels of FVIII range from 50% to
150%. Levels below 50%, or half of what is needed to
form a clot, determine a person’s symptoms. Mild
hemophilia A 5% up to 30% of FVIII in the
blood. People with mild hemophilia Agenerally
experience bleeding only after serious injury, trauma or
surgery. In many cases, mild hemophilia is not
diagnosed until an injury, surgery or tooth extraction
results in prolonged bleeding.
Moderate hemophilia A 1% up to 5% of FVIII in the
blood. People with moderate hemophilia A tend to have
bleeding episodes after injuries; bleeds that occur
without obvious cause are called spontaneous bleeding
episodes. Severe hemophilia A <1% of FVIII in the
blood, people with severe hemophilia A experience
bleeding following an injury and may have frequent
spontaneous bleeding episodes, often into their joints
and muscles (Pavlova and Oldenburg 2013).
Results had been reached in this study showing the
presence of partial deletions in tow brothers patients in
exon 23had been concluded refer to areas that failed to
be amplified by PCR, the sequencieng of this exon
confirm the presence of this tow mutions.
Discussion
The present study is concerned with screening of
some Libyan studied patients with hemophilia A and
the aim of the study was to identify CGA to TGA
mutation by Taq I restriction enzyme in exons 23 , 24 ,
and 26 A+B for the factor VIII (F8) gene that causes
hemophilia A among Libyan patients . Amplification of
areas that contain restriction enzyme sites such as Taq I
with CG dinucleotides in their recognition sequence are
especially useful locations to begin a rapid screeing of
hemophiliacs for mutations. Analysis of the five Taq I
sites in exons 18, 22, 23, 24, and 26 shows that there are
three nonsense mutations in patients HA579, HA360
and HA735 at codons 1941, 2116, and 2209,
respectively (Lavergne et al.1992).
These mutations were C to T transitions changing a
CGA codon for arginine to a TGA termination codon.
The patients all suffered with severe hemophilia
(Lavergne et al.1992). In this study no mutation of three
Taq I sites in coding region of factor VIII gene have an
in-frame CGA codon for arginine. In other studies it
was found that partial deletions involving exons 23, 24,
23-26 and 26 are resulted in severe hemophilia A cases
(Gitschier et al.1986; Youssoufian et al.1987; Lozier
2004). Exons 24-25, 25, 25-26, 23and 24 failed to
amplify in patients. This is the first example of a
moderate form of hemophilia A associated with a
deletion involving two exons (Lavergne et al.1992) in
this study partial deletions for mild hemophilia A in
exon 23 of the factor VIII for two patients of 43 patients
i.e approximately 4.65% of causative mutation in exon
23 failed to amplify in two brother patients.
These data may contribute to the knowledge on the
risk factors of inhibitor development that allows libyan
clinicians to assess whether an individual patient is at
high risk. We performed study of various types of F8
mutations and we obtained more precise estimates of
the relative risks according to the F8 genotypes in
patients with hemophilia A.
Further investigation will be continue this study on
patients from our Hemophilia Treatment Center in order
to reinforce our understanding in the molecular defect
of hemophilia A in Libya and to set up counseling
genetic and the prenatal diagnosis for Libyan families
with hemophilia A. Our study provides more precise
estimates of the relative risks for different F8 genotypes
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Essarbout et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:7-12
. relative to exon 24 in libyan patients with Hemophilia
A. In future research on other possible genetic and
nongenetic risk factors, the predisposition related to the
hemophilic genotype should be taken into account. The
epigenetics study and gene gene interaction may open
new approaches, will facilitate a study about
implication of factor 8 gene in the ethiology of
hemophilia. The design of antagonistic inhibitors of the
factor VIII system (Lozier 2004; Fang et al.2007) could
provide novel anticoagulants for the treatment of
thrombotic disorders (Lavigne et al.2005).
Acknowledgements
We would like to express our sincere and thanks to
the staff of Biotechnology Research Center including
specifically: General director Prof. Dr. Mohamed El
sharief; Head of Genetic Engineering Department; Dr.
Amar Elslughy; Dr. Abdulmunam Abulayha and Eng.
Essam Sharfeddin.
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L .J. V. M. Sci. (2015) Vol. 1 (1): 1-4.
Study of Serotonin Transporters Gene in Sporadic Amyotrophic Lateral
Sclerosis in French Population
Julien Praline1,2
, Refaat M.Tabagh1,4*
, Maïté Amy1,2
, Patrick Vourc’h1,2,3
, Christian Andres1,2,3
and Philippe
Corcia1,2
¹Inserm U930, CNRS 2448, Tours, France,
²Universite´ Francois-Rabelais, Tours, France,
³CHRU de Tours, Laboratoire de Biochimie et BiologieMole´culaire, Tours, France, 4Zoology Department / Faculty of Science / University of Tripoli-Libya.
Received 11 October 2015/ Accepted 18 November 2015
Key words: Amyotrophic lateral sclerosis, 5HTTLPR, serotonin transporter, Genotypes, PCR
Introduction
Amyotrophic lateral sclerosis (ALS) is a fatal
neurodegenerative disorder described worldwide.
During the last 10 years, an extensive literature has
focused on genetic susceptibility factors in populations
from different origins. Currently, there is no universal
genetic factor associated with the majority of ALS
cases.
Amyotrophic lateral sclerosis (ALS), sometimes
called Lou Gehrig's disease, is a rapidly progressive,
invariably fatal neurological disease that attacks the
nerve cells and responsible for controlling voluntary
muscles (muscle action we are able to control, such as
those in the arms, legs, and face) (Kelly 2013). The
disease belongs to a group of disorders known as motor
neuron diseases, which are characterized by the gradual
degeneration and death of motor neurons (Ellison
2008).
ALS is characterized by stiff muscles, muscle
twitching. This results in difficulty speaking,
swallowing and eventually breathing (NINDS 2010).
The cause is not known in 90% to 95% of cases; about
half of these genetic cases are due to one of two
specific genes; it results in the death of the neurons that
control voluntary muscles and the diagnosis is based on
a person's signs and symptoms with testing done to rule
out other potential causes (NINDS 2015). About 5–10%
of cases are inherited from a person's parents (Kim and
Camilleri 2000).
We suggest two ideas the first that neurotransmitters
in particular the serotonin can have a net impact on the
phenotype, functional statue and survival. The second
we suggests that neurotransmitters transporter probably
involved in these modifications both in human and
SOD1 transgenic mice. These results showing the
involvement of serotonin in the rigidity, we propose to
analyses the polymorphism of this gene 5-HTT
serotonin transporter carries neurotransmitter to search
for a possible association between this gene and clinical
parameter ALS patients.
The serotonin is a neurotransmitter existing in both
central nervous systems (CNS) and GI tracts, and
regulates visceral sensation through a paracrine
signaling pathway (Kim and Camilleri 2000). Previous
studies have shown that elevated plasma serotonin is
associated with IBS-D and decreased plasma serotonin
is associated with IBS-C (Atkinson et al. 2006). The
serotonin secreted by enterochromaffin (EC) cells
which not attached to postsynaptic membrane and still
Original article
Abstract Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, invariably fatal neurological disease that attack the
nerve cells and responsible for controlling voluntary muscles. The disease belongs to group of disorders known as motor
neuron disease which are characterized by gradual degeneration and death of motor neurons. Serotonin is a
neurotransmitter existing in both central nervous systems (CNS) and gastrointestinal tracts (GI) and regulates visceral
sensation through a paracrine signaling pathway. Our objective was to study if there is a link between the 5-HTTLPR
(Linked Polymorphism Region) allele of the serotonin transporter gene and the existence of pyramidal or extra-pyramidal
rigidity in different subgroup of ALS patients. This gene has never been studied in ALS. We analyzed the functional
polymorphism 5-HTTLPR of the SLC6A4 serotonin transporter gene in 209 patients with ALS and 214 control
population of 214 individuals. All subjects were of French origin dating at least three generations. The DNA was
extracted from blood cells. The analysis of polymorphism HTTLPR localized in the promoter of the gene of the
transporter of the serotonin (SCL6A4) was performed after PCR in 100ng of DNA. The results are analyzed by agaros gel
electrophoresis (2 %). We obtained tow bands LL genotype with 512pb or the short allele SS with 469pb. We found no
significant differences in allele and genotype frequencies in this 5-HTTLPR polymorphism studied in the SLC6A4 gene
of ALS patients versus control patients. However, further studies will be necessary to fully reject the implication of
SLC6A4in SLA. Indeed the expression of this gene is regulated by epigenetic processes, and these may still be impaired
in SLA. Moreover, effects on mRNA splicing, protein localization, and properties of 5-HTT could be possible
mechanisms for influencing SLA risk.
Libyan Journal of Veterinary and Medical Sciences December 2015 Vol. 1 (2): 13-17
*Corresponding Author: Refaat Mohamed Tabagh, Department of Zoology, Faculty of science, University of Tripoli.
Tripoli, Libya. Tele.+ 218092 404 65 67. E.mail: [email protected].
13
Praline et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:13-17
. in the synaptic cleft, this serotonin will be activated to
reuptake the serotonin back mechanism by the
transporter (SERT) and allow the retune of the
serotonin into enterochromaffin EC cells and activate
the effect of serotonin in GI tracts subsequently
(Bertrand and Bertrand 2010).
Pharmacological and genetic studies also support a
role for serotonin in corticogenesis, and brain
development in general, even prior to the formation of
synapses (Wellman et al 2007). One key regulator of
serotonin levels in the cortex and hippocampus is the
serotonin transporter (5-HTT), which is involved in the
reuptake of extracellular serotonin (Mamounas et al.
2000; Salichon et al. 2001). 5-HTT is a Na+/Cl-
dependant membrane transporter encoded by the
SLC6A4 gene (solute carrier family 6 member 4;
17q11.2) (Figure 1) (Heinz 2005). A polymorphism
(5HTTLPR) in the promoter region of SLC6A4
determines the neuroanatomical size and functional
coupling of the amygdala-frontal cortical circuit in
humans (Pezawas et al. 2005; Watson et al. 2008).
Figure 1: Schematic structure of the human SLC6A4
gene. Non-coding and coding exons are indicated by
white and black boxes, respectively. Locations of the
genotyped polymorphisms and alternative splicing are
indicated.
In present 70% of subjects of European populations
(Heils et al. 1995), it is frequency is lower in African
populations (37%) but nearly augment in Asian 90% in
term of frequency, the second allele (Heils et al. 1995).
Allele 4, 5 and 6 are very rare in Europe (Meyer-
Lindenberg et al. 2005).
Multiple genetics studies have shown a significant
association between the number of repetitions and
neurological disease. In promoter region of SLC6A4
gene the 5HTTLPR polymorphism determines the
neuroanatomical size and functional coupling of the
amygdala-frontal cortical circuit in humans (Heinz
2005). This circuit has been implicated in several
psychiatric disorders including Fragile X and Williams
syndrome, this two pathologies characterized by mental
deficiency (Pezawas et al. 2005).The association
between VNTR polymorphism and psychiatric disease
has been reported several times (Mazer et al. 1997).
These results demonstrate the importance of studying
the frequency of alleles of the VNTR in ALS to
determine if an association exists between the VNTR
genotype and presence of rigidity.
The serotonin transporter (5HTT) is a protein of 630
amino acids of the family Na+ dependent transporters,
with 12 transmembrane domains. The serotonin
transporter gene (SCL6A4) is located 17q11 near
NF1gene and bleomycin hydrolase gene. The SCL6A4
gene consists of 16 exons and extends over 38kb
(Bradley et al. 1997).
Among the described various polymorphisms, some
have functional effects, the polymorphism most studied
are: The insertion-deletion of 44pb in the promoter
region 5` (5-HT Transporter Linked-Polymorphic
Region, HTTLPR). This polymorphism is arepetition of
a member of 20-23pb. The allele contains 14 repeats
(Short S) and 16 repeats (Long L) U 18 repeats (very
long) (Heils et al. 1995). The allele is associated with
increased expression of RNA messanger in vitro (Heils
et al. 1996), And in vivo (Murphy et al. 2004).
A variable number of tandems repeat (Variable
Number of Tandem Repeats VNTR) intron 2 (Lesch et
al. 1994). The allele contains 12 repetitions are more
expressed but this expression also depends on
sequential variations and the tissues were the alleles are
expressed. A version of G to T in the site of
polyadenylation in the UTR3` region (5-HTT3`UTR-
Variant) (Kim et al. 2002), these allelic variant have
been studied in numerous pathology neuropsychiatric
.A lot of articles have been published on the subject last
10 years in particular depression, bipolar disorder,
mood, suicide, autism, and attention hyperactivity
deficit disorder.
SS genotype of 5-HTTLPR is associated with a risk
of depression or not correlate with treatment anti
depression, another neurological disease are studies for
example pulmonary hypertension, the genotype SS is
associated with late onset of Alzheimer disease and the
depression in the Parkinson`s disease (Meyer-
Lindenberg et al. 2005). This study was designed to research if there is association between polymorphism
5-HTTLPR of the gene encoding serotonin transporter
and the ALS phenotype according to weather patients
are rigid (extra-pyramidal rigidity, pyramid or not rigid)
after genotype study by PCR of ALS patients and
control (healthy subjects).
Materials and methods
Currently 209 patients with sporadic defined ALS
and 214 probable or possible controls were included in
this study, Clinical data and blood samples, collected
between 1996 and 2012, were obtained from 209 SALs
patients and 214 controls. We have our DNA bank
which reserved in -20Callowed us to use the DNA bank
for many years and we have a tissue culture of the
patients which reserved in -70 permitting us to use
DNA extraction and use our DNA bank for a long time
(over 100 years). The participants signed consent forms
approved by Medical Ethical Review Boards in French
that adhere to the principles described in the
Declaration of Helsinki. All subjects were of French
origin dating at least three generations. To maintain
comparability between groups, patients and controls
living in France, but of other ethnicity, were excluded
from the study.
The DNA was extracted from blood cells in the
usual methodology (sambrooke et al. 1989),the analysis
of polymorphism HTTLPR localized in the promoter of
14
Praline et al.
Libyan J. Vet. Med. Sci. Vol. 1(2) December 2015:13-17
the gene of the transporter of the serotonin (SCL6A4)
was performed after PCR in 100ng of DNA with 125
μMdNTP, 2% DMSO, 125 μMbuffer 1X, 0,4 U of
DNA polymerase DyNAzyme EXT (Finnzymes) and
200 pmol of every primer : forward, 5HTTF2 : 5’-
GGCGTTGCCGCTCTGAATGC -3’, reverse, 5HTTR2
: 5’-GAGGGACTGAGCTGGACAACCAC-3’. The
conditions of PCR are : 94°C5 min, 35 cycles à 94°C 30
s, 63°C 1 min, 72 °C 1 m in, puis à 72°C 10 min. The
results are analyzed by Agaros gel electrophoresis (2
%). We obtained tow bands LL genotype with 512pb or
the short allele SS with 469pb like show in the gel
electrophoresis photo (Figure 2).
The frequencies of polymorphism between patients
and controls were compared with a chi2 test. All
analysis was performed with JMP statistical software
version 7.0.2 (SAS) Institute, Cary, NC, USA).Allele
and genotype frequencies in patients and controls were
compared with a Chi test x². Odds ratios were estimated
for alleles and genotype (http://www. hutchon. net/
ConfidOR.htm). Linkage disequilibrium (parameter D’)
between markers was calculated using the software
Haploview v.3.32. LD patterns were analyzed by
Spearman rank correlation (Excel) between LD
measures.
Results
The Comparing genotypes and polymorphisms of
HTTLPR between patients and Controls (Table 1 and
2).The HTTPLR polymorphism is not significantly
associated with the Clinic form (bulbar or spinal) ALS
(Table 3).No significant difference was observed
between two groups of patients in the age of onset
(Table 4).
Figure 2: The PCR amplification step allowed differentiating between the short (S,484 bp)
Versus the long allele (L, 528 bp) of 5 - HTTLPR. Gel agar electrophoresis (2 %).
Table 1: The number of chromosomes and allele frequencies of L and
S HTTPLR for the Patients and controls (χ² = 0.02; p = 0.87).
Alleles Frequencies % Number Frequencies % Number
L 58.4 250 57.9 242
S 41.6 178 42.1 176
Total 100 428 100 418
Table 2: Genotypes of HTTPLR polymorphism for the patients and
controls (χ² = 0.02; p = 0.99).
Genotype Frequencies % Number Frequencies % Number
LL 36.5 78 35.9 75
LS 43.9 94 44 92
SS 19.6 42 20.1 42
Total 100 214 100 209
Table 3: Comparison of allele frequencies L and S HTTPLR between two groups
of patients according to the clinical form of the disease (χ² = 2.85; p = 0.09).
Alleles Frequencies % Number Frequencies % Number
L 47.5 56 56.6 172
S 52.5 62 43.4 132
Total 100 118 100 304
15
Praline et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:13-17
.
Table 4: The comparison of allele frequencies L and S HTTPLR between two
groups of patients according to age of disease onset (²= 0.02; p = 0.88).
Alleles Frequencies % Number Frequencies % Number
L 57.4 58 56.5 95
S 42.6 43 43.5 73
Total 100 101 100 168
Discussion and conclusions:
We here report the first genetic study on the
serotonin transporter gene in ALS, We have
investigated functional polymorphism 5-HTTLPR in
these gene in control individuals and ALS patients
matched for origin (Central France) and ethnicity
(Caucasian). Functional studies have reported that the S
allele of 5-HTTLPR was associated with a lower
expression of 5-HTT and a lower serotonin reuptake
activity (Pezawas et al. 2005).
Our results do not support a direct role for the S
allele of 5-HTTLPR in SLA. These preliminary results
concern the gene of serotonin transporter (SCL6A4).
This gene has never been studied in ALS. We did not
find any association between a polymorphism in the
promoter of this gene and ALS. The numbers of
patients are important enough that this conclusion
considered is solid. We also tried to search in different
disease subgroups (sex, age and ethnical groups) if
there were differences in serotonin transporter gene
genotype. No significant difference could be detected,
noted that the bulbar forms appear to be more S alleles
that spinal forms. This would be to check on a larger
population; in fact we do not have the sufficient
statistical power to conclude with certainty.
Wendland et al. (2006) suggested that the effect of
5-HTTLPR on SLC6A4 expression may be due to a
nearby (19 bp) rs25531 polymorphism in the promoter.
The G allele of this polymorphism is located in a
consensus binding site for AP2, a family of
transcription factors described as positive or negative
regulators of transcription (Eckert et al. 2005). Data
from Hu et al. (2000) has shown that the G allele of
rs25531 was associated with a decreased expression of
5-HTT mRNA compared to the A allele.
Serotonergic signaling systems have significant
interactions with overlapping functional targets
(Pezawas et al. 2005). Genetic epistasis between this
system was confirmed by studies on SLC6A4
transgenic mice (Daws et al. 2007). In the present study
on ALS patients, we observed no genetic interactions
between 5-HTTLPR polymorphism studied in SLC6A4.
However, further studies will be necessary to fully
reject the implication of SLC6A4in SLA. Indeed the
expression of this gene is regulated by epigenetic
processes, and these may still be impaired in SLA.
Moreover, effects on mRNA splicing, protein
localization, and properties of 5-HTT could be possible
mechanisms for influencing SLA risk.
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L .J. V. M. Sci. (2015) Vol. 1 (1): 1-4.
The Impact of Wheat Rhizosphere on the Distribution of
Burkholderia spp and Soil Bacteria
Merfat T. Ben Mahmud
Department of Soil and Water - Faculty of Agriculture, University of Tripoli- Tripoli-Libya.
Received 15 October 2015/ Accepted 25 November 2015
Key words: Rhizosphere, Wheat growth stages, Burkholderia, Soil bacteria
Introduction
Plant roots play important roles in shaping microbial
communities in soil by releasing a wide range of
compounds (Garbeva et al., 2008). Although root-
released products comprise of an important pool of
organic compounds for soil micro-organisms, their
composition and quality can vary according to plant
species, soil type and plant development stage
(Chaparro et al., 2013). Due to this variation in
exudation, different plant species growing in the same
soil type were known to select divergent bacterial
communities. However, when analyzing the microbial
community associated with the same plant species
growing in different soil type.
The soil type might exert a great influence on
microbial diversity (Wieland et al. 2001). In view of the
fact that the plant has a large impact on microbial
diversity, one might expect agricultural management to
play an important role as wells (Lupwayi et al. 1998).
Indeed, many agricultural practices, such as crop
rotation, continuous cropping and tillage, induce
changes in the microbial communities in soil (Alvey et
al. 2003; Lupwayi et al. 1998), which may persist long
after the management practice took place. Although
agricultural practices induce general changes in soil
microbial communities, specific microbial groups may
respond differently (Clegg et al, 2003).
A number of studies have reported that the
application of inorganic nitrogen had a significant
impact on Eubacterial and Actinomycete community
structures, whereas soil drainage significantly affected
the community structures of Burkholderia spp and
Pseudomonas spp. In addition, continuous wheat
cropping affected the community structure of
Burkholderia spp such that an increase in the
population of antibiotic producing Burkholderia spp
induced the natural suppression of Take-all disease in
wheat (Raaijmakers and Weller 1998).
Similarly, the establishment of apple orchards in a
field where wheat had previously been grown led to an
decrease of soil suppressiveness against Rhizoctonia
solani, which was correlated with a decrease of
Burkholderia cepacia and Pseudomonads putida
populations (Mazzola, 1999).
Considering the fact that agricultural management
and plant species affect soil microbial communities, the
main objective of this study was to get a better
understanding of how land use and crop species, such
as wheat affect the diversity of the genus Burkholderia.
As well as, this study aimed to address the effect of
the wheat rhizosphere on the distribution of
Burkholderia spp and soil bacteria which factor (land
use) had a greater influence on the soil-borne
populations by the enumeration on plates of rhizosphere
and non rhizosphere soil communities.
Materials and methods
The experiment was conducted in a growth chamber
in Soil microbiology lab at Faculty of Agriculture,
University of Tripoli. Winter wheat (Triticum aestivum
L.) was grown in glass plant tubes (25 x 150) mm were
filled with 20g soil to approximately 5cm from rim. Soil
were collected from two field sites from Wadi Elrabie,
East of Tripoli (Loam sandy soil, pH 8.6) and Janzour,
West of Tripoli (sandy soil, pH 8.1) with three
replicates of each of the two soil samples, giving a total
Original article
Abstract This study was conducted on the effect of the wheat rhizosphere on the distribution of Burkholderia spp. and soil
bacteria communities during wheat plant growth by the enumeration on plates of rhizosphere and non rhizosphere soil
communities in different type of soils, which were collected from different fields, Wadi Elrabie and Janzour (East and
west) of Tripoli, Libya. The results showed that the gram negative species of soil bacteria exceeded the remaining
bacterial population in the rhizosphere on contrast with non-rhizosphere which dominance of gram-positive cocci,
endospore and forming rods. There was no difference in the total bacterial population with plant age , but the types of
colony growth were reversed between the rhizosphere and non-rhizosphere soil in 5 week (seedling stage) and 20
weeks (maturity stage).The genus of Burkholderia prevail in the rhizosphere of the wheat particularly in the early
stages of plant growth (seedling stage). The changes in the trend of the calculated ratio of gram-negative bacteria in
the rhizosphere to that of the non-rhizosphere soil cannot be generalized to all species. Results obtained with
Burkholderia population in this experiment showed different picture.
Libyan Journal of Veterinary and Medical Sciences December 2015 Vol. 1(2): 18-21
*Corresponding Author: Merfat T. Ben Mahmud Department of Soil and Water - Faculty of Agriculture, University of
Tripoli – Tripoli - Libya
Tripoli - Libya. Email:[email protected]
18
Ben Mahmud
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:18-21
. of 24 treatments, which explains three factors: one type
of plant, two soil type (Janzour and Wadi Elrabie soil)
with two sites (rhizosphere and non-rhizosphere soil)
and two stages of wheat growth (seedling and maturity
stage) in three replicate. All tubes were irrigated to field
capacity twice a week. One inoculated seed of wheat
was placed in each tube approximately 1cm below the
surface of the soil.
The bacteria counting were carried out at the end of
the growth cycle which was seedling stage
approximately 5 weeks and maturity stage 20 weeks
after sowing, by using the method of calculating the
number of probabilistic (Alexander 1973). Bacterial
strains were initially isolated from the rhizosphere and
non-rhizosphere soil of wheat roots. Plants were
uprooted from the tubes along with amount of non-
rhizosphere soil.
The non-rhizosphere soil was removed by gentle
shaking of the roots whereas the soil adhering strongly
to the root was referred to rhizosphere soil. Ten gm of
each soil samples were mixed thoroughly in 3 replicate,
then transferred to 250 ml flask with 100 ml sterile
distilled water and were shaken for 30 min at 150 rpm.
Immediately after shaking, a series of 10-fold dilution
in 0.8% (w/v) NaCl solution was prepared for each soil
sample by pipetting 1 ml of aliquot into 9 ml sterile
water. 0.1 ml of each dilution of the series was plated
into a Petri plate with soil extract agar for rhizosphere's
samples and with Dextrose ager for non-rhizosphere's
samples.
Bacteria Burkholderia isolated from soil samples
into plates with Burk’s agar medium.Three replicate
plates were made for each dilution plates were placed in
an incubator at 30o
C for 8 days to estimate
Burkholderia and soil total bacterial population, after
incubation, the plates dilutions of rhizosphere and non
rhizosphere microorganism were Gram-stained using
standard procedures.
Morphology characterisation was determined using
a compound microscope in oil immersion (1000 X)
about 100 colonies were chosen at random at all the
colonies from the rhizosphere whatever their size, shape
and colour were transferred onto other plate to check
for purity. All the colonies grown on the plates were
about 1mm diameter and white with flat margins
initially glossy and gummy but turned into glistening
colonies with clear slime upon further growth. Some of
the species were defined by direct use of microscopic
morphological characteristics and compared to some of
the known and available cultures and then were
characterised using the criteria of Bergey and Holt
(1994).
Results and discussion
This experiment showed the impact of the wheat
rhizosphere on the distribution of Burkholderia spp and
soil bacteria by the enumeration on plates of
rhizosphere and non-rhizosphere soil as shown in table
(1). A difference between the rhizosphere and non
rhizosphere concerned the colonies growth on the
plates. All morphological characteristics conformed to
descriptions of those known from the most of the
rhizosphere colonies of two stages of wheat growth
(seedling and maturity stages) from two soils (Wadi
Elrabie and Janzour). There had a very homogeneous
morphology bacteria rod, ovoid cells treated like
species of Burkholderia, Psedomonas, Azotobacter,
Agrobacterium and endospore as Bacillus sp,
Streptomyces. All colonies had grown after 4 days of
incubation, on the contrast the non rhizosphere soil had
a vast of colonies rod and ovoid, rod forming and
endosperm cells as Streptomyce,s Cellulomonas,
Azotobacter, Cytophaga and Pseudomonas grown after
4 days and some of them grow after 8 days.
Table 1: The common genuses of soil bacteria and their growth on plates listed downward in Wadi Elrabie and
Janzour soils.
Seedling stage
(5 weeks) Maturity stage
(20 weeks)
Rhizosphere
1- Gram negative bacteria, rod, and ovoid cells
and treated like species of Burkholderia,
Psedomonas, Azotobacter and Agrobacterium.
2- Bacteria gram positive, rod and endospore,
Bacillus sp
1- Gram negative bacteria, rod and ovoid
cells. Cytophaqa, Coccobacil,
Azotobater, Burkholderia.
2- Gram positive bacteria, endosperm
forming rod. Bacillus Streptomyces.
Non-
rhizosphere
1- Gram positive bacteria, Cocci, endospore
Staphylococcus, forming rod Streptomyces.
2- Gram negative bacteria, rod Pseudomonas,
Burlholderia and ovoid cells like species of
Azotobacter.
1- Gram positive bacteria. Streptomyces
forming rod Cellulomonas.
2- Gram negative bacteria, rod and ovoid
cells. Azotobacter and Cytophaga.
It can confirm the fact in a number of previous
researches revealed that gram-negative bacteria
population prevails in rhizosphere, while the gram-
positive bacteria population is numerically superior in
non rhizosphere and comes second in rhizosphere
(Macura 1967 and Briwb 1975). It also notes that the
plant age and stages of growth does not affect the
distribution of microbes in the appearance and
disappearance of some specie as a result of the nature of
the root outputs by releasing a wide range of organic
compounds that change from one stage to the other
stages of wheat growth. It had indicated the ratio
19
Ben Mahmud
Libyan J. Vet. Med. Sci. Vol. 1 (2) December 2015:18-21
between the numbers of gram negative bacteria in the
rhizosphere to their numbers in the non-rhizosphere
become even greater with plant age in between stage
seedling and maturity stage (Salles et al.(2006); Ben
Mahmoud 2008).
This study indicates the diversity of the
Burkholderia population (which was a negative-gram
bacteria) associated with rhizosphere was consistently
higher in the rhizosphere than in the non-rhizosphere
There was a greater number of Burkholderia population
at seedling stage (5 weeks ) in rhizosphere than in non-
rhizosphere soil, while thereafter the numbers of
Burkholderia declined to the lowest number at the
maturity stage (20 weeks) were recorded from 7 x 105
to 4 x 104 cfu /gram soil at Wadi Elrabie and from 5 x
104 to 2 x 10
3 cfu /gram soil at Janzour soil. However,
the other bacterial communities’ population of the non
rhizosphere in both soils were increased from seedling
to maturity stages of wheat growth, as shown in table (2
and 3).
Table 2: Total Burkholderia and soil bacteria in wheat rhizosphere and non rhizosphere in
Wadi Elrabie soil
Seedling stage
(5weeks) cfu /gram soil
Maturity stage
(20 weeks) cfu /gram soil
Burkholderia Soil bacteria Burkholderia Soil bacteria
Rhizosphere 7 x 105 4.5 x 10
2 4 x 10
4 8 x 10
3
Non- rhizosphere 5 x 102
6.5 x 104
2 x 102
7 x 105
Table 3: Total Burkholderia and soil bacteria in wheat rhizosphere and non rhizosphere in Janzour soil
Seedling stage
(5weeks) cfu /gram soil
Maturity stage
(20 weeks) cfu /gram soil
Burkholderia Soil bacteria Burkholderia Soil bacteria
Rhizosphere 5 x 104 3 x 10
2 2 x 10
3 7.3 x 10
4
Non- rhizosphere 2 x 102
4.5 x 103 1.2 x 10
2 3 x 10
5
To sum up, the Burkholderia population present in
the wheat rhizosphere higher than in non rhizosphere
(Pallud et al.2001) might be due to the high substrate
availability observed around the roots, indeed the
bacterial communities population of the wheat
rhizosphere seem to have lower level of culturability
than non rhizosphere as proven by Van Elsas et al.
(2002); Salles et al. (2006).
References
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for microbial population, Agronomy Monograph
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Alvey S.. Yang C H., Buerkert A and Crowley D E.
(2003). Cereal/legume rotation effects on
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Ben Mahmud, M., (2008). The effect of Burkholderia as
biofertiliser on cereal productivity. PhD
Dissertation, Biotechnology and Environmental
Biology (School of Applied Sciences) RMIT
University, Melbourne, Australia.
Bergey DH and Holt GJ (1994). Bergey's manual of
determinative bacteriology. Baltimore: Williams &
Wilkins (9th Ed.) 787 pages.
Briwb M E. (1975). Rhizosphere microorganims,
opportunists, bandits or benefactors. Soil
microbiology. Article review Walker, N. pp. 21-38.
Chaparro JM, Badri DV, Bakker MG, Sugiyama A,
Manter DK and Vivanco JM (2013). Root exudation
of phytochemicals in Arabidopsis follows specific
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Clegg C D., Lovell R D L and Hobbs P J. (2003).
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(2008). Rhizosphere microbial community and its
response to plant species and soil history. Plant Soil
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Lupwayi N Z., Rice W A., and Clayton G W.
(1998). Soil microbial diversity and community
structure under wheat as influenced by tillage and
crop rotation. Soil Biol Biochem. 30: 1733-1741.
Macura A. (1967). Physiological studies of rhizosphere
bacteria. In Gray T R. and Parkinson D. The Ecol.of
Soil Bacteria. Liverpool: University. pp.379- 395.
Mazzola M. (1999). Transformation of soil microbial
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potential in response to apple roots. Phytopathology.
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Pallud C., Viallard V., Balandreau J., Normand P.
and Grundmann G. (2001). Combined use of a
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. specific probe and PCAT medium to study
Burkholderia in soil. J Microbiol Methods. 47: 25-
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plant protection by 2,4- diacetylphloroglucinol -
producing Pseudomonas spp. in take-all decline
soils. Mol. Plant Microb. Interact. 11: 144-152.
Salles JF, Van Elsas JD and Van Veen JA (2006).
Effect of agricultural management regime on
Burkholderia community structure in soil. Microb
Ecol. 52: 267–279.
Van Elsas JD, Garbeva P and Salles JF (2002). Effects
of agronomical measures on the microbial diversity
of soils as related to the suppression of soil-borne
plant pathogens. Biodegradation. 13:29-40.
Wieland G., Neumann R. and Backhaus H. (2001).
Variation of microbial communities in soil,
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21
Bushwereb et al.
L .J. V. M. Sci. (2015) Vol. 1 (1): 1-4.
Clinical Outbreaks of Flavobacterium columnare among Tilapia Populations
from Lake Qarun, Egypt
Dina A. Mosselhy1, Mohamed A. El-Aziz
2, Mona M. Husien
1, Abdelsalam A. Abumhara
3 and Alaa E. Eissa
2, 3*
1 Fish Diseases Research Department, Animal Health Research Institute, El- Dokki, Giza, 12618, Egypt.
2 Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Cairo University, 11221, Egypt. 3 Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, University of Tripoli, Tripoli, Libya.
Received 18 October 2015/ Accepted 25 November 2015
Keywords: Flavobacterium columnare, Tilapia, Lake Qarun, Water quality, Experimental infection
Introduction
Lake Qarun extremely affected by a combination of
human activities and climatic changes during the past
5000 years (Flower et al. 2006).The lake suffered
drastic chemical changes for several decades where it
has been used as a general reservoir for agricultural
wastewaters drainage as well as fish farms drainage
throughout Fayoum province (Authman and Abbas,
2007). Fathi and Flower (2005) reported that the
average water temperature of Lake Qarun reached its
maximum in August and September (30 °C) and 16 °C
as minimum value in February. The regular pH of the
lake is an alkaline pH, which has fluctuated between 8.2
in September / October and 9.5 in February.
Tilapias are fast-growing fish which are capable of
surviving in poor water conditions and can tolerate a
wide range of environmental variables (Amal and
Zamri-Saad, 2011). Disease is usually the outcome of
an interaction between the host, the pathogen and
external stressor(s) (Snieszko, 1974). F. columnare
infection usually occurs in fish exposed to poor water
quality (Tripathi et al. 2003). Hansona and Grizzle
(1985) induced Flexibacter columnaris (recently, F.
columnare) infections in channel catfish by exposure to
5 mg/L (ppm) nitrite for 7 days. Jeney and Jeney (1995)
concluded that F. columnare adherence to gill tissues
was enhanced by high nitrite concentration (5 mg/L),
high organic content (2 g/L) and high temperature
(28°C). Tripathi et al. (2003) reported that sudden
changes in water temperature of 5°C or more pose
significant stress, predisposing fish to infection by F.
columnare. Columnaris disease has also been reported
in cold water fish at temperatures ranging from 6 to 12
ºC (Tripathi, 2005). Kubilay et al. (2008) demonstrated
that mortality level in columnaris infected cultured
rainbow trout (Oncorhynchusmykiss) fry, reached
approximately 30% in a day when water temperature
increased to 16°C.
F. columnare is an opportunistic pathogen in which
temperature rise above 20 ºC enhances the bacterial
pathogenicity and virulence by enhancing bacterial
proteolytic enzymes secretion. Thus, increased
pathogen virulence together with sharp decrease in
dissolved oxygen due to temperature rise will
jeopardize the immune system of fish and increases the
potential of the ubiquitous bacterial invasion (Eissa et
al. 2010).
Members of the genus Flavobacterium are widely
distributed in soil and water. Flavobacterium species
induced diseases among fresh water and marine fish
(Husien, 1999). The taxonomy of F. columnare has
been changed several times over the years basis on
morpho-chemical criteria. The old taxonomical names
of the pathogen included, Chondrococcus columnaris,
Cytophaga columnaris and Flexibacter columnaris
(Bernardet and Grimont 1989). The most recent name
“Flavobacterium columnare” was approved by the
microbiological community after molecular typing of
the worldwide archived strains (Bernardet et al. 1996).
F. columare has been isolated from several fish species
worldwide such as Nile tilapia (O. niloticus) (Husien,
1994; Husien, 1999); fingerling and adult Nile tilapia in
Brazil (Figueiredo et al. 2005); Nile tilapia (O.
niloticus) and Nile catfish (Clarius gariepinus) in
Lower Egypt (Eissa et al. 2010); juvenile rainbow trout
in the southern part of Turkey (Kubilay et al. 2008),
some tropical fish species in Brazil (Pilarski et al.
2008).
Kubilay et al. (2008) discovered that in wet mount
Original article
Abstract During multiple clinical outbreaks that happened between the fall and winter of 2012. A total of 59 fish pathogenic
bacteria were retrieved form 150 clinically diseased Tilapia species collected from different regions through Lake Qarun.
The dermotropic yellow pigmented F. columnare have represented 3.4% of the retrieved Flavobacterial isolates. The
main clinical findings exhibited by F. columnare natural infection were deep ulcerations and tail rot. The lake’s poor
water quality such as alkaline pH , high levels of un-ionized ammonia, high nitrite concentrations, together with the
abrupt rise of water temperature have played a potential role in triggering F. columnare infection. To assess the
pathogenicity of the retrieved isolates, experimental infection was carried out by intraperitoneal injection of O. niloticus
with F. columnare isolates and mortalities were detected within 10 days. F. columnare isolates were sensitive to
Danofloxacin, Nalidix acid, Nitrofurantoin and Oxolinic acid while they were resistant to Amoxicillin, Cephalothin,
Gentamicin, Lincomycin, Oxytetracycline, Ampicillin, Tetracycline.
Libyan Journal of Veterinary and Medical Sciences December 2015 Vol. 1 (2): 22-28
*Corresponding Author: Alaa E. Eissa. Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, University
of Tripoli, Tripoli, Libya. P. O. Box 13662 Tripoli, Libya. Tele.+218 911571033, Email: [email protected].
22
Mosselhy et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:22-28
. Preparation of F. columnare showed a slow gliding
movement and characteristic column-like masses. F.
columnare isolates are positive for oxidase, catalase,
flexirubin pigment, H2S production, nitrate reduction
and gelatinase tests. Most of isolates grew on Shieh
agar supplemented with tobramycin with the
development of yellow rhizoid colonies. F. columnare
do not utilize carbohydrates. Growth can occur at 0.5%
NaCl but not in 1% NaCl and no growth occurred at
4°C. Eissa et al. (2010) have presented a full morpho-
chemical profile for F. columnare which included
positive catalase; chromo-shift after adding potassium
hydroxide 3 % onto the bacterial colonies on Hsu-
Shotts agar plates; Congo red binding ; gelatin
hydrolysis; growth at 25 ºC, 30 ºC and 37 ºC; growth in
the presence of Neomycin sulfate and Polymyxin B
sulfate.
Materials and methods
Fish sampling
A total number of 170 apparently healthy and 150
clinically diseased Tilapias were collected alive or
freshly dead from different localities of Lake Qarun
during the period from October 2011 to December 2012
during the course of three emergent outbreaks. The
fishes were kept on ice until transferred to the Fish
Diseases Research Laboratory for further processing.
Clinical and post mortem examination of naturally
infected fish were carried out according to the methods
described by Stosckopf (1993).
Isolation and purification of F. columnare
Skin and gills chunks from examined fish were
taken by sterile curved forceps and streaked directly
onto Hsu-Shotts agar medium (Bullock et al. 1986)
supplemented with 0.5 and 1 % NaCl (El-Nasr
pharmaceutical chemicals, Egypt). The inoculated
plates were incubated at 25ºC for 5-7 days. Presumptive single colonies from the achieved pure cultures were
picked and streaked onto different laboratory media:
Tryptic soya agar (TSA, Merck, Germany) with 3%
NaCl, MacConkey agar (Oxoid, England) and TCBS
agar (Oxoid, England).
Colonial and morphological characters
Colonial and morphological characters of F.
columnare were studied according to Austin and Austin
(2012).
Biochemical identification of F. columnare
Bacterial isolates were presumptively identified
using both cultural characteristics and conventional
biochemical tests recommended by Griffin (1992).
Biochemical tests include catalase test, adding
potassium hydroxide 3 % onto the bacterial colonies on
Hsu-Shotts agar plates, Congo red binding (by flooding
24 h cultures with 5 mL of Congo red dye), gelatin
hydrolysis, growth on TSA, growth at 25 ºC, 30 ºC and
37 ºC , growth at 0.5 % , 1 %, and 3 % NaCl. Motility
was detected by stabbing of the single colony from the
pure isolates into semisolid agar. Further identification
of F. columnare isolates was adopted by API® 20 E and
NE; according to the instructions of the BioMérieux
Company.
Water analysis
Collection of water samples for physico-chemical
parameters testing was conducted according to APHA
(1998). Water samples were collected concurrently with
fish samples from Shakshouk and Deir-El Birka drains
at Lake Qarun basin. Each sample was collected from
the mid zone of water source and at a suitable depth in a
sterile dry 500 mL glass bottle for chemical
examination.
Water temperature was measured by using floating
ordinary thermometer (Model Heto T-08- china), while
pH was measured using pH meter (Model 25025-
Jenway-Germany). Ammonia and nitrite were
determined using Colorimeter (Model Jenway 6030-
Germany) at wavelength 603 and 545 nm respectively.
Experimental infection
F. columnare was grown on Hsu-Shotts agar
medium with 0.5 % and 1% NaCl. The pure cultures
were harvested by cotton swab and suspended into
sterile saline according to Austin and Austin (2007).
A total of 30 O. niloticus with average weight (55±2
g) were used in the experiment. The fish were divided
into two groups. First group containing 15 O. niloticus
were divided into 10 fishes (I/M injected with F.
columnare at a dosage of 0.5 ml of 107 CFU/mL) and 5
fishes (S/C injected with the same dose) according to
the method described by Husien (1994). . The second
group (control group) comprises 15 O. niloticus that
were similarly divided into 10 fishes (I/M injected with
0.35 mL saline) and 5 fishes (S/C injected with the
same dose). The injected fish were kept under
observation for 10 days post challenge during which
mortalities, clinical signs and PM were recorded on a
daily basis. Koch’s pathogenicity postulates were
fulfilled by re-isolation of the infective pathogen for
verification of the specificity of death.
Antibiogram
The disc diffusion method was used as described by
Bauer et al. (1966).
Results
Incidence of natural infection of F.columnare among
examined Tilapia populations
F. columnare prevalence among naturally infected
Tilapia spp. was (3.4%) among the positive cases of
fishes (39) for the different retrieved pathogens. The
seasonal prevalence of F. columnare was recorded
100% in winter.
Clinical findings in naturally infected fishes
The clinical signs of the F. columnare naturally
infected fish included deep skin ulcerations, tail rot and
hemorrhage at the base of pectoral fins (Figure 1.A and
B). On postmortem examination, infected Tilapias
exhibited pale necrotic gills and enlarged and congested
liver.
23
Mosselhy et al.
Libyan J. Vet. Med. Sci. Vol. 1 (2) December 2015:22-28
Figure 1: Naturally infected Tilapia spp., (A) showing tail rot. (B) showing deep ulceration on the
operculum under the eye.
Bacteriological examination
Morpho-chemical characteristics of retrieved
isolates
The colonial characters of retrieved isolates on solid
media were yellow rhizoid colonies adhering to the
Hsu-Shotts agar surface after 5-7 days of incubation
(Figure. 2. B). No growth was obtained on TSA 3%
NaCl after 7 days of incubation, also no growth on
MacConkey agar or TCBS agar. Gram stained smears
(Figure. 2. A) from pure colonies revealed the presence
of long Gram-negative bacilli. These morphological
characteristics were presumptive for F. columnare.
Biochemical identification of the retrieved isolates
using conventional biochemical tests
F. columnare were identified using a panel of
biochemical tests listed in table (1).
Further identification of the retrieved isolates by
API® 20 E and NE
The retrieved isolates were further biochemically
confirmed using the semi-automated API® 20 E and
NE. Isolates identities were confirmed through the API®
20 E and NE software matching system with an
accuracy percentage exceeding 98 % .
Intensity of F. columnare infection in skin of
examined fishes
The intensity of infection (The percentage of F.
columnare isolation) from skin of naturally infected
Tilapias was 100%.
Water quality assessment
The assessed pH of water samples was alkaline while
un- ionized ammonia, and Nitrite concentrations
exceeded the permissible limits (Table 2).
Figure 2: (A) Gram- negative long bacilli of F. columnare. (B)Yellow rhizoid colonies
of F. columnare adhering to Hsu-Shotts agar surface after 5-7 days of incubation.
24
Mosselhy et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:22-28
.
Table 1: Biochemical characteristics of F. columnare
Biochemical test Result
Oxidase +ve
Catalase +ve
Gram stain -ve long bacilli
KOH 3% Gm –ve (mucoid)
KOH 20% Chromoshift to pink-brown colonies
Congo red binding +ve and at washing with distilled water, no removal
of colonies
Indole -ve
Methyl- Red -ve
Voges-Proskauer -ve
Citrate -ve
Triple Sugar Iron Alkaline/Alkaline
Hydrogen sulfide production +ve
Glucose -ve
Sorbitol -ve
Sucrose -ve
Maltose -ve
Urease -ve (weak +ve)
Gelatin liquefaction -ve
O/F test glucose +/-
Growth at 4oC Slight growth after 48 hours incubation
Growth at 37oC No growth after 48 hours incubation
Growth on TSA +3% NaCl No growth
+ ve = Positive, -ve = Negative, O/F = Oxidation/ Fermentation, +/- = Positive/ Negative
Table 2: Results of water quality assessment
Locality
Water parameter
Shakshouk Deir-El Birka drain References
Temperature 19 ºC 22 ºC -
pH 8.51 7.55 -
Un-ionized toxic
Ammonia 0.0534 mg/L 0.0126 mg/L
Upper Limit 0.0125
mg/L,(Swann, 1997)
Nitrite 0.02 mg/L 0.02 mg/L 100 µg/L, (Ali, 2002)
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Mosselhy et al.
Libyan J. Vet. Med. Sci. Vol. 1 (2) December 2015:22-28
Experimental infection
A 60 % of O. niloticus I/M injected with F.
columnare were dead within 6 days post-infection and
40% of those S/C injected were dead within 6 days
post infection. It is worthy to mention that mortalities
were recorded on a daily basis throughout the 10 days
period of the experiment. The clinical signs and post
mortem examination of experimentally infected fishes
is illustrated in figure (4).
Antibiogram
Antibiograms of the presumptively identified F.
columnare revealed that all isolates were sensitive to
Danofloxacin, Nalidix acid, Nitrofurantoin and
Oxolinic acid. All isolates were also resistantto
Amoxicillin, Cephalothin, Gentamicin, Lincomycin,
Oxytetracycline, Ampicillin, Tetracycline and
Clamoxyl, while intermediate resistance was exhibited
against Colistinsulphate and Trimethoprim /
Sulphamethoxazole (Figure 5).
Figure 4: (A) O.niloticus experimentally infected (S/C) with F. columnare, Showed deep hemorrhagic ulceration on the
trunk. (B) O. niloticus experimentally infected (I/M) with F. columnare showed hemorrhagic musculuature, enlarged
and congested internal organs.
Figure 5: (A) F. columnare with intermediate resistance to CT and resistant to OT, CN,
AMX, MY. (B) F. columnare sensitive to F, OA, DFX and resistant to AML and AMP.
Discussion
The sharp environmental changes such as sharp
fluctuations in temperature, pH and dissolved oxygen
are highly incriminated as predisposing factors for
initiating F. columnare infection (Wakabayashi, 1991
and Tripathi et al. 2003). The fluctuations in water
temperature above 16 up to 25 ºC then 30 ºC is known
to enhance the bacterial pathogenicity and virulence by
increasing the bacterial growth rate; increases the
adhesion capacity of the bacterium to the fish tissues
(Decostere et al. 1999); activates the Chondrotin AC
lyase (Griffin, 1992) which degrades polysaccharides,
particularly those found in cartilaginous connective
tissue (Teska, 1993).
The sharp decrease in dissolved oxygen due to
temperature rise has suppressed the immune system of
fish with increasing the potentials of the ubiquitous
bacterial invasions (Amend, 1983; Bullock et al. 1986;
Suomalainen et al. 2005). Further, the increased
ammonia levels with consequent rise in water pH are
the most possible triggering factors for initiation,
establishment and spread of infection (Sniezko, 1974;
Holt et al. 1975; Morrison et al. 1981; Bullock et al.
1986; Suomalainen et al. 2005). Despite the fact that
early summer and early autumn are the flaring up
seasons for F. columnare (Eissa, 2010), yet, the
seasonal incidence of F. columnare was 100 % in
winter season, which is in partial agreement with
Peselis (2011) who concluded that Columnaris disease
could be enhanced when the fishes are in stressed
environments.
The skin ulcerations fin and gill rot can be mainly
attributed to Chondroitin AC lyase which is an enzyme
produced by F. columnare, that has the capability to
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Mosselhy et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:22-28
. degrade polysaccharides, particularly those found in
cartilaginous connective tissue (Eissa et al. 2010).
The results of morpho-chemical testing of the
retrieved isolates were in full agreement with the
standard criteria of F. columnare described by Austin
and Austin (2012), Eissa et al. (2010) and Peselis
(2011). Controversially, the ability to grow at 4 ºC in
which slight growth took place after 48 hours of
incubation is against data described by Kubilay at al.
(2008) who detected no growth at such temperature. F.
columnare was 100% isolated from skin which is
compatible with the findings of Tripathi et al. (2005)
who declared that F. columnare was readily detected in
skin specimens from infected fish. However, the
bacterium was infrequently detected in liver, kidney and
spleen samples and suggested that Columnaris disease
generally occurs as a cutaneous disease that is
unassociated with systemic infection.
In respect to experimental infection of O. niloticus
with F. columnare, 60% mortality within 6 days of
(I/M) and 40% (S/C) injections were obtained. These
results relatively agreed with Husien (1994) who
reported 100 % mortalities within 5 days post S/C
injection of O. niloticus. Clinical signs and post mortem
examination of experimentally infected fishes were
almost similar to those of naturally infected fishes. This
confirmed that the recorded clinical and PM findings in
naturally infected fishes were due to Flavobacteriosis.
Ultimately, the antibiogram results of the confirmed
F. columnare isolates were consistent with similar data
recorded by Husien (1999) who reported complete
resistance to Tetracycline and Ampicillin. However, the
antibiogram data disagreed with Hawkeab and Thuneab
(2011) who mentioned that all F. columnare tested
strains were susceptible to Terramycin (Oxytetracycline
HCl).
Acknowledgments
Authors are grateful to all members of the
Department of Fish Diseases and Management, Faculty
of Veterinary Medicine, Cairo University and Fish
Diseases Research Department, Animal Health
Research Institute for facilitating the use of laboratory
facilities throughout the entire study.
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Bushwereb et al.
L .J. V. M. Sci. (2015) Vol. 1 (1): 1-4.
Effects of Mercury Chloride on Sperm Parameters and Pathological Changes in
Kidney of Mouse
Habiba A. El Jaafari1*, Amr M.Fathalla
1 and Suhera Aburawi
2
1Department of Zoology, Faculty of Science, University of Tripoli, Tripoli-Libya. 2Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, University of Tripoli, Tripoli-Libya.
Received 20 October 2015/ Accepted 25 November 2015
Key words: Mercury Chloride, Sperm, Mouse, Kidney
Introduction
There are a serious rise in the environment of toxic
chemicals and heavy metals. The accumulation of
heavy metals in the body of organisms causes
significant health risks. Mercury is one of the fairly
harmful heavy metals for lives (Gul et al. 2004) and it
has extremely toxic characteristic for organisms
(Sharma et al. 2005). Mercury is a naturally occurring
metal in organic (methyl mercury, ethyl mercury and
phenyl mercury) and inorganic forms (mercury
chloride) which possess different toxicity (Trasande et
al. 2005). The potential health effects of mercury have
been a matter of concern because of potential wide
human exposure consequent to its wide spread use.
It distributed throughout the environmental by both
natural sources and human activities and easily
accumulated in the animal tissues. Most of the mercury
in the atmosphere is elemental mercury vapor and
inorganic form, which may be deposited in water, soil
and sediments. Mercury vapor is highly lipophilic and
is efficiently absorbed through the lungs and oral
mucosa (Crinnion 2000). Mercury is widely used in
refinery, plastic and paints antiseptic, scientific
instruments, photography, fuel combustion, wastewater
treatment facilities, paper mills and medical and
agriculture field.
This metal easily penetrates into tissues such as
kidney, lung, blood stream, connective tissue, brain,
adrenal and other endocrine glands (ATSDR 1999;
Joseph et al.2001; Jagadeesan and Sankarsamipillai
2007) and it may be caused damages in all these tissues.
Reproductive hazards from metal exposure in males are
one of the fastest growing areas of concern in
toxicology today. Exposure to different heavy metals
causes irreversible toxic insult to male reproductive
system. Heavy metals produce cellular impairments at
structural and functional level in male reproductive
system. The effect of heavy metals, such as lead,
mercury, cadmium, chromium and arsenic on male
reproduction has been studied in details in various
experimental species.
Metals could interfere with the gametogenic cells or
Leydig cell or spermatozoa directly in semen. These
effects may results in reduced fertility or associated
with pregnancy wastage, congenital malformation
associated with genetic diseases. Potential toxicity of
Metals caused alteration in sperm morphology, count,
motilityas well as biochemical disruptions of enzymes
and hormones (Jagadeesan and Sankarsamipillai 2007).
Therefore, the present study aims to evaluate the effects
of mercuric chloride on sperm parameters and
histopathology on kidneys.
Materials and methods
Animals and Diet
Forty Swiss albino male mice 8 to 10 weeks of age
weighing 28-30 g were used for this study. These mice
were inbred in the animal house of the Zoology
Department/ Faculty of Science / University of Tripoli.
The mice were housed in plastic cages containing
wooden flakes and had free access to water and
standard diet (ad libitum).
Experimental design
Experiment was conducted for eight weeks. Animals
were divided into four groups, each group contain ten
animals. Group I was received an equivalent amount of
Original article
Abstract
Mercury chloride (HgCl2) is a toxic substance increasingly being recognized as a potential environmental pollutant.
In this study, sperm parameters have been studied to relate the effects of HgCl2 on mice fertility and to evaluate the
toxicity of HgCl2 on kidney. The kidney is well documented as the target organ for HgCl.2. Forty Swiss albino male
mice, eight to ten -week-old and weighing 28 - 30 g. Animals were divided into four groups, each group contain ten
animals and injected intraperitoneally weekly with 0, 0.5, 1.0 and 2.0 mg mercury chloride /kg body weight for eight
weeks respectively. Sperms of vas deferens were evaluated with respect to sperm count, motility, and morphology.
Total sperm count and motility were significantly decreased in dose dependent manner of Mercury chloride, while
sperm abnormality increased consistently at different doses of mercurial treatment over a period of eight weeks.
Maximum sperm abnormality among the treated groups was noted in the group given 2 mg/kg body weight. The
study revealed that Mercury chloride has a potency to alter sperm parameters. Also, histological alterations were also
observed in the Kidney.
Libyan Journal of Veterinary and Medical Sciences December 2015 Vol. 1 (2): 29 -32
*Corresponding Author: Habiba A. El Jaafari, Department of Zoology, Faculty of Science, University of Tripoli, Tripoli-
Libya, Tele: 0927227431, E. mail: [email protected] , Department of Zoology, Faculty of science, University of Tripoli. Tripoli, Libya. Tele.+ 218092 404 65 67. E.mail:
29
El Jaafari et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:29-32
. distilled water and served as control. Group II, Group
III and Group IV were injected intraperitoneally with
0.5, 1.0 and 2.0 mg mercury chloride /kg body weight /
weekly respectively.
Seminal Fluid collection
Groups of treated and untreated mice were killed
after 24 hours from the last injection by cervical
dislocation. Sperm of each mouse were obtained by
squeeze the vasa deferentia gently into 1ml normal
saline in small dish. The specimen was mixed gently
by a special dropper to distribute the seminal fluid.
Sperm suspension was incubated for 15 minutes at 32
ºC to allow sperm separation (El Jaafari 1995).
Determination of sperm count:
Sperm counts were made using the method by
Sakamoto and Hashimoto (1986) modified by El Jaafari
(1995). The sperm were counted by charging both
chambers of improved Neubauer Hemocytometer with
sperm suspension. Sperm count expressed as million
per millileters.
Examination of sperm morphological
For sperm morphology test, two smears were made
from each mouse, and allowed to dry in air. Smears
were stained with 1% eosin Y in water for 10 minutes.
From each mouse 500 sperms were examined at 400
magnifications for morphological abnormalities.
Results were expressed as percentage of abnormal
sperm (Otitoloju et al. 2010).
Determination of sperm motility
Sperms from both treated and untreated mice were
examined according to Ficsor and Ginsberg (1980) by
using the improved Neubauer hemocytometer
(American optical Co., Buffalo. N. Y). Numbers of
motile and non-motile sperms of treated and untreated
mice were counted under the X40.
Histological study
For histological examination, specimens from
kidneys tissue were taken immediately after sacrificing
mice, and fixed in 10% formalin solution. The fixed
specimens were then trimmed, washed, dehydrated, and
embedded in paraffin. Sections of 5 µm thickness were
cut with a manual microtome, stained with Hematoxylin
and Eosin (H&E). The stained sections were examined
under the microscope and the different cell types were
carefully studied and photographed (Bancroft and
Gamble 2002).
Statistical analysis
The statistical analysis was conducted using SPSS
(Software packing version 18). Treatment was
compared by applying one way ANOVA. Post hoc test
(LSD) was performed. The difference was considered
significant at p<0.05.
Results
The effect of mercuric chloride (HgCl2) on sperm
motility, count and abnormality is shown in (Table 1).
The results indicated that the administration with HgCl2
caused a significant (p≥0.05) decrease in both sperm
counts and sperm motility with associated significant
(p≥0.05) increase in the percentage of abnormal sperms
in dose dependent manner as compared with the control
group. Sperm morphology test of treated mouse with
different doses of mercuric chloride showed different
sperm phenotype abnormalities such as Club-shaped
head, Banana-like head, bent head, fused head, two
tails, lasso-like and folded mid piece.
Table 1: Toxic effect of mercuric chloride on sperm count, motility and morphology of mouse.
Treatments Sperm count
(106/ml)
Number of motile
sperm(106/ml)
Number of sperm with
abnormal morphology
Control 26.15 ± 0.33 10.87 ± 0.35
(41.56%)
11.00 ± 1.72
(2.2%)
Mercuric chloride
(0.5mg/kg) 20.9 ± 0.29*
7.00 ± 0.37*
(33.49%)
63.37 ± 3.47*
(12.67%)
Mercuric chloride
(1.0mg/kg) 17.52 ± 0.62*
3.62 ± 0.49*
(20.66%)
115.62 ±5.04*
(23.12%)
Mercuric chloride
(2.0mg/kg) 13.45 ± 0.43*
3.62 ± 0.49*
(26.91%)
171.62 ± 10.28*
(34.32%)
Parameters= mean± SE; *, significantly different compared to control mice at p≤0.05.
Numbers in parenthesis represent the percentage value.
Histopathological observations:
HgCl2 administration caused prominent
histopathological damage in kidneys compared with
those of control group mice (Figure 1). The kidney
sections from the control group preserved histological
structures with normal appearance of glomerulii,
proximal and distal convoluted tubules and collecting
tubules (Figure 1.A). In contrast, the kidney sections of
HgCl2 treated mice with 0.5mg/kg and 1.0 mg/kg
showed significant areas of tubular dilation with
hemorrhage and degeneration of renal corpuscles,
inflammation of renal epithelium, congestion of renal
lumen and cloudy swelling (Figure 1.B and Figure 1.C).
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El Jaafari et al.
Libyan J. Vet. Med. Sci. Vol. 1 (2) December 2015:29-32
The kidney treated mouse with 2.0 mg/kg showed
hydropic degeneration of renal epithelial tubules
(Figure 1.D).
Figure 1: Histopathological changes in kidney of mouse: (A) photomicrographof untreated mouse kidney (control), (B)
Photomicrograph of treated mouse kidney with 0.5 mg/kg Hgcl2 showed renal tubular dilations with hemorrhage and
degeneration of renal corpuscles, inflammation of renal epithelial and congestion of renal lumen, Cloudy swelling, (C) Photomicrograph of treated mouse kidney with 1.0 mg/kg Hgcl2, (D) Photomicrograph of treated mouse kidney
with 2.0 mg/kg Hgcl2 showing hydropic degeneration of renal epithelial tubules.
Discussion
The mercuric chloride toxic effect is due to its
ability to forms organomercury complexes with proteins
(Lorshieder et al.1995). It has great affinity for thiol-
groups of Biomolecules, such as glutathione (GSH)
and sulfhydryl proteins, which may contribute to its
toxicity (Hansen et al.2006). In the present study the
administration of different doses (0.5, 1.0 and 2.0
mg/kg) of HgCl2 caused significant (p≤0.5) reduction in
both sperm count and sperm motility with associated
significant increase in the percentage of abnormal
sperms as compared with the control groups, which
assure with those results obtained by other investigators
(Otitoloju et al. 2010 and Rao and Sharma, 2001).
These changes may be attributed to impairment of
sperm maturation and secretory functions of epididymal
cells which might be due to oxidative stress or
insufficiency of androgens. It was also in agreement
with Khan et al. (2004) which reported that mercuric
chloride produced adverse effects on reproductive
performance of mice even though the route of
administration was different. Moreover, it was
consistent with the investigation of Fossato da silva et
al.(2011). Which relate the disturbance in sperm
parameter (increase in sperm abnormal shape and
decrease in both sperm motility and total sperm count )
to the effects of mercuric chloride on testicular
spermatogenic and steroidogenic functions in
experimental animals.
It is well known that mercuric chloride is one of the
pro-oxidants that induce oxidative stress which cause
lipid peroxidation and membrane damage leading to
loss sperm motility (Vachhrajani et al.1988). Mercury
can concentrate in the kidneys and testes, leads to
kidney failure and infertility through the effects on
epididymis and interfere in spermatogenesis (Eto et
al.1997), these facts might be the explanation for the
decrements in sperm count, motility and morphology in
this study.
Histopathological examination revealed that mice
administered with different doses of mercuric chloride
shows disturbance in structural integrity and cloudy
swelling compared with control group, it is well known
that the mercuric chloride accumulate more in renal
epithelium, so the endothelial damage by reactive
oxygen species could be the main sources for producing
large scale hemorrhages in kidney ,liver ,brain and
heart. Degenerative changes in the renal tubules
epithelium of kidneys may be due to deposit of
mercuric chloride in the kidneys, this results was in
agreement with other investigators (Oda et al.2012 and
Gado et al.2014). The pathological changes in the
kidney tissue include interstitial oedema, tubular
dilation, and sloughing of individual epithelial cells.
Also administration of mercury chloride dose 2.0 mg
/kg body weight causes hydropic degeneration in renal
epithelium.
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El Jaafari et al.
Libyan J. Vet. Med. Sci. Vol.1 (2) December 2015:29-32
. Conclusion:
On the basis of sperm parameters, and kidney
pathological feature, our finding suggest that exposure
to mercuric chloride in male Swiss albino mice causes
severe tissue damage in all segments of kidneys and has
different effects on sperm parameter in short time. It
becomes clear that mercuric chloride exhibit
considerable toxic effect on most vital organs. So there
are urgent needs to develop preventive measures to
contain mercuric chloride toxicity.
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