LIST OF ABBREVIATIONS Abbreviation Meaning

158 Pages·2012·2.2 MB·English

LIST OF ABBREVIATIONS
Abbreviation Meaning
CATT Card agglutination test for trypanosomiasis
cDNA Complementary Deoxyribonucleic acid
D.W. Distilled water
DEPC Di ethyl pyro carbonate
DNA Deoxyribonucleic acid
dNTPs Deoxyribonucleotide triphosphate
EDTA Ethylene diamine tetra acetic acid
ELISA Enzyme-linked Immunosorbent Assay
EtBr Ethidium bromide
GRP Glucose Regulated Protein
Hsp Heat Shock protein
IPTG Isopropyl – β- D- thiogalactoside
kDNA Kinetoplastid Deoxyribonucleic acid
LB Luria- Bertani
LMP Low Melting Point
mRNA Messenger ribonucleic acid
NCBI National Center for Biotechnology Information
PAGE Polyacrylamide Gel Electrophoresis
PBS Phosphate buffer saline
PCR Polymerase chain Reaction
PK Buffer Proteinase K Buffer
PK Enzyme Proteinase K Enzyme
PVDF Polyvinylidene fluoride
R. T. Room temperature
RE Restriction enzyme
RNA Ribonucleic acid
RNase Ribonuclease
RMSD Root-mean-square Deviation
RPM Revolution per minute
SDS Sodium dodecyl sulphate
SOC Super Optimal Broath with Catabolite
Repression
TAE Tris- Acetate EDTA
TE Tris EDTA
UV Ultra Violet
X- gal 5 -bromo-4-chloro-3-indolyl-beta-D-
galactopyranoside
Units of Measurement
% Percentage
µg Microgram
µl Microlitre
0C Degree Celcius
A Absorbance
bp Base pair
cm Centimeter
Da Dalton
g Gram (s)
h Hour (s)
IU International unit
Kbp Kilo base pair
KDa Kilo Dalton
Kg Kilogram
M Molar
ma Milli ampere
mg Milli gram
min Minute (s)
Min Minute
ml Milli litre
mm Mili meter
mM Milli molar
ng Nano gram
OD Optical density
pmol Picomole
sec Second (s)
U Unit
V Volt
V / cm Volt per centimeter
V / V Volume / volume
W / V Weight / volume
APPENDICES

APPENDIX – I

1. Agarose Gel Electrophoresis buffer

1.1. TAE buffer (50x)
Stock solution:
Tris base 121 gm
Glacial acetic acid 28.55 gm
0.5 M EDTA acid (pH 8.0) 50 ml
Water upto 500 ml
Working concentration of TAE buffer (1x)
TAE buffer 10 ml
Water 490 ml
1.2. TE (Tris/EDTA) Buffer
Tris-HCl (pH 7.5) 10 mM
EDTA 1 mM
Make from 1M stock of Tris-HCl (pH 7.5) and 500 mM stock
of EDTA (pH 8.0).
Working solution:
1M Tris-HCl (pH 7.5) 1.0 ml
500mM EDTA (pH 8.0) 0.2 ml
Water to 100 ml
2. Phosphate Buffered Saline buffer (1X), pH 7.4
NaCl 8.0 gm
Na HPO .2H O 1.44 gm
2 4 2
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KH PO 0.24 gm
2 4
KCl 0.20 gm
Distilled water to make 1000 ml
3. Trypanosome separation buffer (PSG buffer, pH 8.0)
3.1. Solution A
Na HPO (anhydrous) 8.000 gm
2 4
NaH PO .2H O 0.780 gm
2 4 2
NaCl 4.250 gm
Distilled water to make 1000 ml
3.2. Solution B
Glucose solution dextrose 10 gm
Distilled water to make 400 ml
Just before use 6 parts of solution A was mixed with 4 part of
solution B
4. Proteinase K buffer
Tris base (ph 8) 100mM
EDTA 10mM
NaCl 50mM
SDS 2%
β Mercapto ethanol 20mM
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APPENDIX – II

1. Giemsa stain
Giemsa stain 1 ml
Distilled water 9 ml
Slides were stained for 45 minutes.

2. 6X loading dye
Bromophenol blue 0.25%
Xylene cyanol 0.25%
Glycerol 50%
EDTA 2 mM

3. Ethidium bromide solution (10 mg/ ml)
Ethidium bromide 0.2gm
Sterile Water 20 ml

4. Agarose (0.8%)
Agarose 800 mg
TAE buffer 100 ml

5. Agarose (1.2%)
Agarose 1.2 gm
TAE buffer 100 ml

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APPENDIX – III

1. Phenol: Chloroform: Iso amyl alcohol; 25:24:1
Phenol 250 ml
Chloroform 240 ml
Iso amyl alcohol 100 ml

2. Alcohol (70%)
Alcohol 70 ml
Water 30 ml

3. Luria Bertani (LB) Medium ( 500 ml)
Tryptone 1.0% (5 gm)
Yeast extract 0.5% (2.5 gm)
NaCl 0.5% (2.5 gm)
Adjust the pH to 7.0 with NaOH
For LB plates, add 1.5% (7.5 gm) agar to the LB broth and
autoclave.

4. Luria Bertani (LB) Agar (500 ml)
Tryptone 1.0% (5 gm)
Agar 1.5% (7.5 gm)
Yeast extract 0.5% (2.5 gm)
NaCl 0.5% (2.5 gm)
Adjust the pH to 7.0 with NaOH

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5. LB plates with ampicillin/ IPTG/ X- Gal
LB plates with ampicillin were made by adding ampicillin to a
final concentration of 50µg/ml after cooling of LB agar to
500C. Then 100 µl of 100mM IPTG and 20 µl of 20mg/ml X-
Gal was spreaded over the surface of the LB- ampicillin plate
and allowed to absorb for 30 minutes at 37ºC prior to use.

6. SOC medium (100ml)
Tryptone 2 gm
Yeast extract 0.5 gm
NaCl (1M) 1 ml
KCl (1M) 0.25 ml
Mg+2 stock (2M) 1ml
Glucose (2M) 1ml

2M Mg+2 stock (100ml)
MgCl 6H O 20.33 gm
2. 2
MgSO . 7H O 24.65 gm
4 2

7. X-GAL solution
X-GAL 20mg/ml
Dissolved in 100% N,N dimethyl formamide.
20 µl X-GAL was used for 25-30ml LB agar medium in LB
plate.

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8. IPTG solution
IPTG 100mM
Dissolved in distilled water.
100µl IPTG was used for 25-30ml LB agar medium in LB
plate.

9. Ampicillin solution
Ampicillin 50µg/ml
Dissolved in distilled water as 10mg/ml stock.
To make 25ml of LB broth medium containing 50µg/ml
Ampicillin, 125 µl of 10mg/ml ampicillin stock was used.
For 500 ml LB agar 2.5 ml of 10 mg/ml ampicillin stock was
added, ensure that LB agar cooled to 50oC before adding
ampicillin.

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1. INTRODUCTION
Trypanosomosis caused by Tryponosona evansi is an important
haemoprotozoan disease of domesticated animals, pets and wild
animals. It is commonly termed as Surra in all animal species and
“Tibersa” in camels. Trypanosoma evansi has the widest geographical
range of all the pathogenic trypanosome species and the host species
principally affected are camel, horses and dogs. Among the several
species of trypanosomes, Trypanosoma evansi is the most commonly
occuring species in India causing the disease. For non-prevalence of
other species, non-availability of the vector is the main cause. The
disease results in anaemia, lowering down of milk yield and working
capacity. Hence, besides causing economic losses, it also causes high
mortality in valuable animals. Animal trypanosomosis is now a days
considered as a permanent constraint for livestock productivity in
Africa, Asia and Latin America with their geographical distribution still
evolving (Desquesnes et al., 2013).
Surra is a five component system including hosts, protozoa,
environment, vector and reservoir. The causative agent, Trypanosoma
evansi, an intercellular haemoflagellate protozoan is transmitted
mechanically by haematophagous flies like Tabanus, Lyperosia and
Stomoxys genera and is endemic in Africa, Asia and South America
The haematophagous flies, including Tabanids and Stomoxys, are
implicated in transferring infection from host to host, acting as
mechanical vectors. In Brazil, vampire bats are also implicated in a
unique type of biological transmission.
The clinical signs of surra are indicative but are not sufficiently
pathognomonic, thus, diagnosis must be confirmed by laboratory
methods (Dia et al., 1997). The disease in susceptible animals,
including camels (dromedary and bactrian), horses, buffalo, cattle and
pigs is manifested by pyrexia, directly associated with parasitaemia,
together with a progressive anaemia, loss of condition and lassitude.
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Such recurrent episodes lead to intermittent fever (as high as 44°C in
horses (Manohar et al., 1984; Gill, 1977) and parasitaemia during the
course of the disease. Oedema, particularly of the lower parts of the
body, rough coat in camels, urticarial plaques and petechial
haemorrhages of the serous membranes are sometimes observed. In
advanced cases, parasites invade the central nervous system (CNS),
which can lead to nervous signs (progressive paralysis of the hind
quarters and, exceptionally, paraplegia), especially in horses, but also
in other host species before complete recumbency and death.
Abortions have been reported in buffaloes and camels
(Gutierrez et al., 2005; Lohr et al., 1986) and there are indications that
the disease causes immunodeficiency (Dargantes et al., 2005., Onah
et al., 1998). There is considerable variation in the pathogenicity of
different strains and the susceptibility of different host species. The
disease may manifest as an acute or chronic form, and in the latter
case may persist for several months, possibly years. The disease is
often rapidly fatal in camels and horses, but may also be fatal in
buffalo, cattle, llamas and dogs, however these host species may
develop mild or subclinical infections. Wild animals such as deer,
capybara and coati can become infected and ill (including death), but
they may also constitute a reservoir. Animals subjected to stress –
malnutrition, pregnancy, work – are more susceptible to disease.
Biologically T. evansi is very similar to T. equiperdum, the causative
agent of dourine (Brun et al., 1998, Claes et al., 2005), and
morphologically resembles the slender forms of the tsetse-transmitted
species, T. brucei brucei, T. b. gambiense and T. b. rhodesiense.
Most of the molecular characterisations indicate that various
strains of T. evansi isolated from Asia, Africa and South America are
very homogeneous and may have a single origin (Ventura et al., 2002),
but other works suggest that T. evansi could have emerged from T.
brucei in several instances (Jensen et al., 2008; Lai et al., 2008).
Molecular characterisation using random amplified polymorphic DNA
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