Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
65
COMPARING THE EFFECT OF MODIFICATION INNNNCULTURE
MEDIUIM FOR LEISHMANIA
SPECIESUSINGDIFFERENT MAMMALS
Nada Noori* , Amina N.AL-Thwani** ,KhdhrMawla***
* ,**Institute of genetic engineering and biotechnology for post graduat studies,
University of Baghdad,Baghdad<Iraq
***Ministy of Health-Baghdad,Baghdad,Iraq
Keywords:Leishmania,Culture,NNN medium,Horse blood
ABSTRACT
The efficacy of the in vitro cultivation of promastigotes of threeLeishmania spp. were
tested inmodified the biphasic Novy-MacNeal
Nicolle (NNN) medium prepared using blood from different mammals ( human blood
group O , horse, donkey, goat and sheep).
This study was carried out to determine which modification of NNN media will be
gave the best yield in the shortest time fordifferent parasite species, in order to obtain
a large crop of promastigotes for experimental work and for antigen preparation.
Promastigotes of the three main parasite species ,Leishmaniadonovani,
Leishmaniatropica and Leishmania major, isolated from patient in Bagdad and
cultuerd, at equal numbers, in the 5 different NNN preparations. At the end of the 7th
day, the NNN medium using horse blood produced the greatest number of
promastigotes for all Leishmania spp. tested, 27 ,15 ,33x106 whilst goat blood provid
the poorest medium, providing culture results only for L. donovani,7x106.Human
blood group O give good results, 9.2 ,5,4X106.Donkey blood 6.5,2 ,1 X106.Then
Sheep blood gave 4.5, 2.5 X106 ,This finding may be explained by the fact that
Leishmaniais a nicotinamide adenine dinucleotide (NAD) auxotrophand horse
erythrocytes support NAD-dependent microorganisms
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
66
INTRODUCTION
More than 30 Leishmania species (Protozoa, Trypanosomatidae)are known
worldwide, 21 of which may be transmitted to humans by the bite of infected female
phlebotomine sandflies (Diptera, Psychodidae) (1) causing leishmaniasis. Even
though Leishmaniases are found in 98 countries worldwide (2), they are classified as
Neglected Tropical Diseases (NTD) (3). Since the spread of the parasite and its
vectors to new areas, due to factors like climatic changes and human activities,
pathogenicity and drug resistance as well as for the development of new drugs. Most
of these studies require a huge number of parasites, which are produced by cultivation
in vitro using different culture media (4).
The most common culturemedium used for parasite isolation from biological
samples andsandflies is the biphasic Novy-MacNeal-Nicolle (NNN). Yet, it is not
always easy to produce a good number of promastigotes in a short time, for all
species, something vital in research regarding many aspects of the parasite and the
disease.
NNN medium, usually prepared using rabbit or sheep blood is onsidered effective
for the isolation of parasites from biological
Samples although often fails to produce a great number of promastigotes in a short
time (5). in order to develop a medium which would be the most suitable for the
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
67
isolation of the largest possible number of strains in order to be practical and not to
miss out strains that are new in the lab/country/area.
MATERIAL AND METHODS
Parasites
Amastigote of, Leishmania major,.andLeishmaniatropica were used in thisstudy,
isolated from patient with a cutaneous lesion newly introduced during an
epidemiological survey conducted in Baghdad,while
L. donovani was isolated from a patient with visceral disease from bon marrw
,cultured in NNN media at 26Co ± 1 Co. The development of promastigotes was
checked every 2 days( 6).Promastigotes were used for inoculation on day three after
the second passage(15).
Blood
Blood samples from four different mammals - donkey, horse,sheep and goat e were
used to prepare the NNN biphasic culturemedium. Blood originating from four to five
animals of each species were used for the preparation of each kind of NNN in order to
avoid factors related to animal's health condition which may give misleading
results.All animals were healthy and originated from non-leishmaniasis endemic
regions(7).
The blood samples were mechanically defibrinated under sterile conditions,using a
cone-shaped flask containing glass beads which was shaken at low speed ,for 20 min
,at room temperature (8)
Preparation of NNN medium
For the preparation of the NNN culture medium: 16 g/L blood agar, D-glucos 8
g/L, antibiotic gentamycin 1.5ml, 900 mL dH2O wereadded.The mixture in a glass
flask and autoclaving at 121Co for 30 sec , after which the defibrinated animal blood
(150 ml/L) was added. The NNN tubes were prepared and kept at 4 _C for at least 2
days before they were used for parasite inoculation.Prepar liquid phase (loks
solution)(8).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
68
Inoculation and incubation of promastigotes
Each of the four Leishmania strains were inoculated into three
tubes of each different NNN culture medium. The inoculum consisted of 0.5 ml,
containing 200,000 stationary-phase promastigotes. They were incubated at 26 ± 1 Co
for o total period of 2 months. The proliferation potentialof the parasites in the NNN
culture media containing blood of fivedifferentmammela was assessed on day 7, day
14 and then checkedfor live parasites at the end of month 1 and month 2. All cultures
were performed in triplicate. The experiment was repeated twiceand the results were
confirmed.
Light microscopy
To estimate the parasite population on day 7 and day 14, 0.1 ml
of the liquid medium in the NNN tube (nearly all liquid phase of the culture). The
number of parasites per ml was estimated by countingthe parasites using a Thomas
Slide after immobilizing the promastigotes with 10% formaldehyde, to check the
viability of the parasites so only live parasites were taken into account (9).
Statistical Analysis
The Statistical Analysis System- SAS (17) program was used to effect of
difference group in study parameters . Least significant difference –LSD test
was used to significant compare between means in this study.
RESULTS
After 7 days of culture, all three Leishmania spp. developed only in the NNN
medium containing horse blood; which provided, by far, the best crop compared to
the others tried (Table 1).Human blood group O produced cultures lower numbers
than horse blood. Donkey and sheep blood produced cultures, in lower numbers and
for some species they required longer than 7 days to appear. Goat blood NNN
exhibited the poorest results providing parasites only of L. donovani; the other species
remained negative after two months of culture. L. donovani developed in all four
NNN mediums tried, but provided best yield in horse NNN whilst L. Tropica culture
became positive after 14 days in NNN using donkey and sheep blood. The
proliferation ratio of L. major in horse blood NNN was 2.1 times higher to that of L.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
69
tropica, 1.9 to that of L. donovani (Table 1). The positive cultures kept producing
Leishmania parasites after two months. Horse blood NNN was successfullyused for
parasite culture three months after preparation.Horse blood (defibrinated)
.Significance was defined as P ≤ 0.05.
Table 1:The proliferation potential of threeLeishmania spp. in five different NNN
media using blood of different mammela, during a seven day period. Initial inoculum
in all cases .200,000 stationary -phase promastigotes.
NNN
media
L.donovani
X106/ ml
L. tropica
X106/ ml
L. major
X106/ ml
Horse 27±2.04 15± 0.77 33± 2.09
Human blood
group O
9.2± 0.83 5± 0.65 4± 0.06
Donkey 6.5± 0.52 2± 0.05 1± 0.008
Sheep 4.5± 0.30 2.5± 0.07 -a
Goat 7± 0.44 - -
LSD value 4.0528* 3.603* 4.794
*CP<0.05
aPromastigotes appeared after 14 days of culture. The cultures which werenegative (-)
remained negative after two months of culture..
DISCUSSION
The NNN culture medium is widely used for isolating andculturing Leishmania
parasites for many clinical and laboratorypurposes. It is easy to prepare, can be kept in
the freeze for over a month before use, if necessary, and it costs very little to make.
The most common NNN culture medium used is prepared using sheep blood, which
can be obtained commercially. The only drawback of this culture medium is its
weakness to provide good, healthy parasitesof many different species, in a short time
(10).This study was carried out prepare an NNN culture medium which would be
suitable for the three main Leishmania spp. found in the Old World, that is: L.
donovani, L. tropica and L. major; the first two causing visceral and the other two
cutaneous disease.The horse blood NNN proved the most suitable for all three
Leishmania spp. and yielded, by far,
The highest number of parasites after only one week incubation. Human blood
group O NNN was the secondmost efficient allowing the production of promastigotes
of threeLeishmania species. in 7 days and the fourth after 14 daysof culture; but it
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
70
provided considerably lower numbers compared to the horse blood NNN (Table
1).Donkey and Sheep blood NNN preparation allowed the propagation of all
3Leishmania spp. but after 7 days for. L. tropica, and 14 days for L. donovani and L.
major; and in considerable lower numbers compared to horse blood. Goat blood
allowed only the growth of L. donovanipromastigotes , which appeared 7 days
afterinoculation, but the other parasite species did not grow in thismedium, even after
2 months.From our findings it appears that horse blood is the most suitable of the ones
tested in providing a high number of parasites in a short time for all three Leishmania
spp.
Horse blood is relatively easy to obtain in large quantities in most countries,
compared to other smaller animals as rabbits whose blood is also used for the
preparation of NNN culture medium. Rabbit blood is collected either from a rabbit's
ear artery or from the animal's heart.
The blood fromthe ear of one rabbit is enough to make only two to four NNN
tubes (depending on the size of the animal), whereas it is usually a terminal procedure
taking the blood using cardiac puncture(16).Different Leishmania spp. are found in
different geographical regions; and some areas are endemic for more than one
species.Therefore it is useful to have a culture medium such as horse blood NNN in
which most, if not all, species can grow. Such a medium can help in the isolation of
parasites for a rapid diagnosis of visceral andcutaneous disease; but also in isolating
strains of newly introduced species of Leishmania in an area or even species
circulating unnoticed since the culture medium used cannot sustain them.
For a culture medium to be favorable to an organism, it mustmeet its requirements for
development and proliferation. Theability of horse blood to provide this, to all species
tested, may be explained by the fact that Leishmania is a nicotinamide adenine
dinucleotide (NAD) auxotroph organism (11) and since horse erythrocytes support
NAD-dependent microorganisms and lack measurable NADase (12), the enzymes that
participate in NAD metabolism, they may provide what is essential for parasite
growth.
Nicotinamidase plays a key role in Leishmania's cellular development as it requires
assimilating NAD precursors (nicotinamide or vitamin B3, nicotinic acid,
nicotinamideriboside) from the host environment to synthesize NAD by a salvage
pathway (11). Nicotinamidase is a key enzyme of this salvage pathway that catalyses
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
71
conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher
eukaryotes. The enzyme has been shown to be important for growth and proliferation
of L. donovani and essential for establishing an infection (13).
Goat blood,which contains a thermo labile inhibitor, an NAD inactivating enzyme, may
prevent the growth of NAD-dependent microorganisms by depleting available pyridine
nucleotides. Earlier work has shown that goat, bovine and sheep erythrocytes (14), all of
which inhibit the growth of NAD - requiring organisms, like the bacteria Haemophilus
spp., possess high NADase activity (12). Conversely, the erythrocytes of horse, guinea
pig, and rat support Haemophilus species growth and lack measurable NADase. A level
of about 40 times the concentration of NAD present in 5% chocolatized sheep blood agar
is needed to propagate Haemophilus spp. on unheated sheep blood agar (12). Similarly,
fragility of rabbit erythrocytesin agar plates results in gradual release of their NAD and
NADPcontents into the medium. Due to high NADase and negligibleNADPase activity of
rabbit red blood cell stroma at neutral pH, the NAD released into the medium is
hydrolyzed and NADP remains intact (14).
الخاص لاجناس اللشمانیا بأستعمال دم NNN مقارنة التأثیر لتحویر الوسط الزرعی
لمختلف اللبائن
ندى نوری یونس* ،امنة نعمة الثوینی** ،خضر مولى العبادی***
*،**معھد الھندسة الوراثیة والتقنیات الاحیائیة للدراسات العلیا،جامعة بغداد ،بغداد ،العراق
***وزارة الصحة ،دائرة صحة بغداد الکرخ ،بغداد ،العراق
الخلاصة
لوسط ثنائی الطور NNN فعالیة المزروع المسوط فی المختبر بوسط محور
حصان ،حمار ، ماعزوالغنم) وکان الھدف ،(O) اعدت باستخدام الدم من الثدیات المختلفة (فصیلة دم الانسان نوع
اعطى افضل عائد فی اقصر وقت لانواع الطفیلیات المختلفة من اجل الحصول على NNN ھو اختبار ای وسط
محصول کبیر من الطور المسوط لاجل التجارب وتحضیر المستضدات .لثلاث انواع من اللشمانیا نوعین جلدیة
عزلت من المرضى المصابین بالقرحة الجلدیة ونوع احشائی عزل من نخاع العظم لمرضى من بغداد ثم زرعت
باعداد متساویة فی خمسة من الاوساط المحورة المحضرة ،فی نھایة الیوم السابع وجد باستخدام وسط دم احصان
ینتج اکبر عدد من الطور المسوط لجمیع انواع اللشمانیا المستخدمة بالاختبار، 27,15,33 بینما دم الماعز ثبت انھ
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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افقر وسط فقط اعطی نتائج لطفیلی اللشمانیا الحشویةفقط، 7، ودم الانسان نوع () اعطى 9.2 و 5و 4 ثم دم الحمار
اعطى 6.5 و 2و 1.دم الاغنام اعطى 4.5 و 2.5 .ھناک تفسیر حقیقی ان اللشمانیا وکریات دم الحصان تکون غنیة بھذه
تحتاج نیکوتینماید ادنین دانیوکلیوتاید (NAD). المادة
REFRENCES
1- Kaye, P.,and Scott, P., (2011). Leishmaniasis: complexity at the hostepathogen
interface. Nat. Rev. Microbiol. 9, 604-615
2- Alvar, J., Velez, I.D., Bern, C., Herrero, M., Desjeux, P., Cano, J., Jannin,
J.,and den Boer, M.,(2012). Leishmaniasis worldwide and global estimates of its
incidence. PLoS One 7 (5), e 35671.
3- Pawar, H., Renuse, S., Khobragade, S.N., Chavan, S., Sathe, G., Kumar, P.,
Mahale, K.N., Gore, K., Kulkarni, A., Dixit, T., Raju, R., Prasad, T.S., Harsha,
H.C., Patole,andM.S.,Pandey, A., (2014). Neglected tropical diseases and omics
science: proteogenomics analysis of the promastigote stage of Leishmania major
parasite. OMICS 18 (8), 499-512
4- Al-Bashir, N.T.,andRansam, M.B., (1992). Axenic cultivation of amastigotes of
L. donovani and L. major and their infectivity. Ann. Trop. Med. Parasitol 86, 487-
502.
5- Sundar, S.,andRai, M.,(2002). Laboratory diagnosis of visceral Leishmaniasis.
Clin. Diagn. Lab. Immunol. 9 (5), 951-958
6- Messaritakis, I., Mazeris, A., Koutala, E.,and Antoniou, M., (2010).
Leishmaniadonovanis.l.: evaluation of the proliferation potential of promastigotes
using CFSE staining and flow cytometry. Exp. Parasitol. 125 (4), 384-388.
7- Parasuraman, S., Raveendran, R.,andKesavan, R.,( 2010). Blood sample
collection in smalllaboratory animals. J. Pharmacol. Pharmacother. 1 (2), 87-93
8-Wilson, J.D.,and Grimes, A.J., (1968). Defibrination of normal human blood for
in vitro cell studies. Nature 218Q, 178-180.
9- Dutta, S., Ray, D., Kolli, B.K.,and Chang, K.P.,( 2005). Photodynamic
sensitization of Leishmaniaamazonensis in both extracellular and intracellular stages
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
73
with aluminumphthalocyanine chloride for photolysis in vitro. Antimicrob. Agents
Chemother. 49 (11), 4474-4484.
10 - Limoncu, M.E., Balcio_glu, I.C., Yereli, K., Ozbel, Y., Ozbilgin, A.,(1997). A
new experimental in vitro culture medium for cultivation of Leishmania species. J.
Clin. Microbiol. 35 (9), 2430-2431.
.
11- Michels, P.A.,andAvil_an, L., (2011). The NAD metabolism of Leishmania,
notably the enzyme nicotinamidase involved in NAD salvage, offers prospects for
development of anti-parasite chemotherapy. Mol. Microbiol. 82 (1), 4-8
12-Gratten, M., Battistutta, D., Torzillo, P., Dixon, J., Manning, K., (1994).
Comparison of goat and horse blood as culture medium supplements for isolation and
identification of Haemophilusinfluenzae and Streptococcus pneumoniae from upper
respiratory tract secretions. J. Clin. Microbiol. 32 (11), 2871-2872
13-Gazanion, E., Garcia, D., Silvestre, R., G_erard, C., Guichou, J.F., Labesse,
G., Seveno, M.,Cordeiro-Da-Silva, A., Ouaissi, A., Sereno, D.,Vergnes, B.,(2011).
The Leishmanianicotinamidase is essential for NAD production and parasite
proliferation. Mol. Microbiol. 82 (1), 21-38.
14-Artman, M.,andFrankl, G., (1982). Nicotinamide adenine dinucleotide and
nicotinamide adenine dinucleotide phosphate splitting
enzyme(s) of sheep and rabbit erythrocytes: their effect on the growth of
Haemophilus. Can. J. Microbiol. 28(6),696-702.
15-Ntais, P., Christodoulou, V., Tsirigotakis, N., Dokianakis, E., Dedet, J.P.,
Pratlong, F.,and Antoniou, M.,( 2014). Will the introduction of Leishmaniatropica
MON-58, in the island of Crete, lead to the settlement and spread of this rare
zymodeme? ActaTrop. 132, 125-130
16-Sadigursky, M.,andBrodskyn, C.L., (1986). A new liquid medium without blood
and serum for culture of hemoflagellates. Am. J. Trop. Med. Hyg. 35, 942-944.
17-SAS .(2012).Statistical Analysis system ,User"sGuide.Statistical .Version 9.1thed
.SAS. Ist.Inc.Cary .N.C.USA
