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Outline Of Research Work Seminar
Cloning and silencing of Subolesin, Cathepsin L and
Calreticulin genes of Hyalomma anatolicum anatolicum
and evaluation of cross-protective efficacy of
recombinant protein(s)
1
Dr. Binod KumarRoll No.1374Ph.D. ScholarDivision Of ParasitologyIVRI
INTRODUCTION
Ghosh et al., 2005,2007
106 species106 species
4
TICKS
Major tick species in INDIA infesting livestock
Hyalomma anatolicum anatolicumCattle, Buffalo and small ruminantsThree hosts tick
Rhipicephalus (Boophilus) microplusCattle, Buffalo, Horse, donkeys, goat, Sheep, Deer, Pig, Dog, and some wild animalsOne host tick
Direct and Indirect effects
Tick attachmentDeep bite woundAnnoyancePredispose to myasisReduced hide value
Tick secretions Tick toxicosisTick paralysisTransmission of tick borne pathogens
Blood feedingAnemiaProduction and reproduction losses
ControlHigh cost of acaricide treatmentEcological damageHuman health
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Vectorial capacity
H. a. anatolicumTheileria annulata
T. buffeli
T. lestocardi
CCHF (????)
R. (B.) microplus
Babesia bigemina
B. bovis
Anaplasma marginale
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Economic impact of tick infestation on livestock industry
De Castro, 1997;
Minjauw and Mc
Leod, 2003;
Dayton et al., 1991;
Horn, 1987;
Mukhebi et al., 1999
* * Control cost
**
In million US $
8
Methods of Control Major Control Method
Side effects of chemical control method
Chemical control (Acaricides)
Biological control
Genetic transformation
Immunological control
Herbal formulation as
acaricide
Genetically resistance breeds
Acaricides
Selection of resistant
ticks
Environmental pollution
Residues in livestock
products
High cost of repeated
application
Development of new
generation acaricide
Sustainable control ????????
Ticks control strategies
Immunological control (component of IPM)
Immunization with crude antigen
Immunization with Purified Native antigen
Immunization with recombinant antigen
1986- Bm86 identified
1994s- Bm86 based vaccine TickGARD™ and Gavac™ introduced in
market
Reduction in number of application of acaricides
Reduction in incidence of Tick borne disease
Variable efficacy of vaccine against different strain of R. (B.) microplus
(40% - 90%)Cross protective efficacy was poor except R. (B.) annulatus
de la Fuente et al., 2007
Homologue of Bm86 was identified in different ticks species and efficacy was recorded in the range of 40-60% (Liao et al., 2007; Odongo et al., 2007; Kamau et al., 2011; Nijhof et al., 2010)
In India, cross-protective efficacy of Bm86 and its homolog, Haa86 in Hyalomma anatolicum anatolicum was recorded
Antigen Ticks Efficacy Reference
Haa86 H. a. anatolicum 46-80% Azhahianambi et al., 2009; Jeyabal et al., 2010; Kumar et al., 2012
Haa86 R. (B.) microplus 36% Kumar et al., 2012
Bm86 H. a. anatolicum 25% Kumar et al., 2012
Bm86 R. (B.) microplus 44% Kumar et al., 2012
Attachment to host
Salivary gland products like
Cement protein (Kemp et al., 1982)
Anti-haemostatics (Sauer et al., 1995)
Vasodilators, anti-inflamatory , immunosuppressive factors (Champagne, 1994)
Digestive system
Molecules involve in blood meal digestion (Lara et al., 2005)
Iron metabolism (Horn et al., 2009)
Gut associated molecules (Williadsen, 2004)
Haemocoel
Transporter molecules (de la Fuente et al., 2010)
Other molecules involved in physiology of ticks
Major areas of target
Targets selected for study
CalreticulinAnti-thrombotic and complement-inhibition activities in host
Cathepsin LPart of a gut-associated multi-peptidase complex. Its endopeptidaseactivity is important in the initial phase of haemoglobinolysis.
Subolesin(Expressed in all organs and tissues)Function as transcription factors in the regulation of gene expression
Objectives
1. Cloning and sequencing of Subolesin, Calreticulin and Cathepsin L
genes of Hyalomma anatolicum anatolicum and Rhipicephalus
(Boophilus) microplus
2. Evaluation of Conservation of target genes in different isolates of H. a.
anatolicum and R. (B.) microplus
3. Characterization of target genes of H. a. anatolicum
i). Through RNA interference
ii). In-vivo immunization trial using recombinant protein(s)
.•.
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REVIEW OF LITERATURE
Native proteins as Vaccine targets
Immunogen Challenge dose Percentage protection
Larvae Nymphs Adults Immediate rejection (%)a Overall decrease in successive stageb
Reduction in egg massesc
AFF-TLE (L), 39kDa 4000 200 - 60.0 (L),44.0 (N) 34.0 (N),43.2 (A)
Aff-GHLAg (Gut), 34 kDa)
2000 140 40 pairs 24.2 (L),22.4 (N),32.2 (A) 31.2 (N), 25.2 (A), 15.0
Aff-HNAg (Nymph, 39kDa
1600 140 40 pairs 38.0 (L), 25.0 (N), 32.2 (A) 32.7 (N), 28.7 (A), 20.0
Aff-GHAAg (A), 68kDa 2000 140 - 10.3 (L) 17.6 (N) -
HGLA (L), 34 kDa 2000 - 25 pairs 39.0 (L), 28.0 (A) 16.0 (N), 15.8
HGLA (L), 34 kDa 3000 - 75 pairs 32.0 (L), 23.4 (A) 29.32 (N), 50.67
GHLgP (Gut), 37kDa - 140 - 17.0 (N) 20.7 (A) -
aa Mean differences in immediate rejection between immunized and control groups of animal. Mean differences in immediate rejection between immunized and control groups of animal.bb Mean differences in the development of successive stage of the ticks fed on immunized and control group of Mean differences in the development of successive stage of the ticks fed on immunized and control group of animals.animals.cc Mean differences in the egg masses laid by the ticks fed on immunized and control group of animals. Mean differences in the egg masses laid by the ticks fed on immunized and control group of animals.
* p < 0.01.* p < 0.01.
** p < 0.05** p < 0.05Indian J. Exp. Biol., 1998, 1999; Trop. Anim. Hlth. Prod., 2003; J. Parasitic Dis., 2003; Indian J. Exp. Biol., 1998, 1999; Trop. Anim. Hlth. Prod., 2003; J. Parasitic Dis., 2003;
Trop. Anim. Hlth. Produ., 1999; Trop. Anim. Hlth. Produ., 2001, ; Exp. Appl. Acarol., 2000; Trop. Anim. Hlth. Produ., 1999; Trop. Anim. Hlth. Produ., 2001, ; Exp. Appl. Acarol., 2000; Indian J Anim. Sci., Exp. Appl. Acarol., 2003, Parasitology Res., 2005, Trop Anim Hlth Indian J Anim. Sci., Exp. Appl. Acarol., 2003, Parasitology Res., 2005, Trop Anim Hlth Prod., 2005; J Vet Parasitol., 2007; Vaccine 2008Prod., 2005; J Vet Parasitol., 2007; Vaccine 2008
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Recombinant proteins as Vaccine -
Bm86 and its homolog
TickGARD™, Gavac™
20-30% reduction in engorge tick number
30% reduction in engorge tick weight
60-80% reduction in egg masses
50-60% reduction in number of acaricide treatment in a year (Willadsen et al., 1995, de la Fuente et al., 2007)
S.N. Tick Strain Efficacy (E%)
1 Camcord 72-91
2 Yeerongpilly 75
3 Cenapa 84
4 Tuxpan 51
5 Mora 58
6 Colombian field 60
7 Brazilian field 51
8 Argentinain strain 0
Variable efficacy of Bm86 based vaccine against different strain of R. (B.) microplus
de la Fuente et al., 1995, 2005, 2006; Garcia-Garcia et al., 2000
The use of Bm86 based vaccine on cattle tick strains located in different
geographic areas has presented variable efficacy, even the so-called vaccine
failures that seemed to be due to variations in amino acids in the protein codified
by the Bm86 locus (de La Fuente and Kocan, 2003)
de la Fuente et al. (2000) characterized at the molecular level R. (B.)
microplus strains ( 10 strains) from Latin America and Australia,
employing sequences derived from the Bm86 coding region (base
No. 1646 to 1752)
Results
1% nucleotide variation within strains
7.5% nucleotide variation between strains
These variation cause change in amino acid composition at four places
Sossai et al. (2005) collected thirty R. (B.) microplus strains from various geographic regions of Brazil, Argentina, Uruguay, Venezuela and Colombia were analyzed for the Bm86 gene
Gene amplified and sequenced from 278–1071 base (794 base pairs)
Variations from 1.76 to 3.65% were detected in the nucleotides sequence
and 3.4–6.08% in the amino acid sequence of the Bm86 protein
Garcia-Garcia et al. (1999) suggested that variations greater than 2.8% in the amino acid sequence of the protein expressed would be sufficient to confer vaccination inefficiencies when recombinant antigens are used.
Cross-protectionVaccine molecules
Tick species Effect Reference
Bm86 Hyalomma dromedarii DT%-27; DR%- 31; DO%- 32
Rodríguez-Valle et al., 2012
Bm86 Amblyomma cajennense No effect Rodríguez-Valle et al., 2012
Bm86 R. (B.) annulatus E%-99% Canales et al., 2009
Bm86 R. (B.) decoloratus DT%- 45; DR%- 55; DO%- 61
Odongo et al., 2007
Bm86 Rhipicephalus sanguineus Reduction in Larvae, Nymph and Adults, 38%, 29% and 31%, respectively
Perez-Perez et al., 2010
Bm86 R. appendiculatus No effect de Vos et al., 2001
Bm86 Hyalomma anatolicum anatolicum
E%- 25 Kumar et al., 2012
Bm86 Homolog
Ree86, Dr86, Hm86, Av86, Ir86, Os86 (Nijhof et al., 2010), Hl86 (Liao et
al., 2007), Ra86 (Kamau et al., 2011), Ba86 (Canales et al., 2008), Bd86
(Odongo et al., 2007), Rs86 (Fang and Xu, 2007) and Haa86
(Azhahianambi et al., 2009)
Efficacy of some of recombinant protein was evaluated which is variable
Antigens Tick species Efficacy Reference
Ba86 R. (B.) annulatus 83% Canales et al., 2009
Ba86 R. (B.) microplus 71% Canales et al., 2009
Haa86 H. a. anatolicum Larvae – 47- 60%Adults - 40-80%
Azhahianambi et al., 2009; Jeyabal et al., 2010; Kumar et al., 2012
Haa86 R. (B.) microplus 25% Kumar et al., 2012
Some other recently identified Vaccine targets-
Targeted molecules Species of Ticks Experimental animal
Vaccine Efficacy Reference
Acid phosphatase (HL-3)
41.0 kDa
Haemaphysalis longicornis Rabbit DR% = 10.6
Mortality (%) = 28.0
Zhang et al., 2011
Hc-23
43 kDa
Haemaphysalis concinna Rabbit DR% = 11
DO% = 62
Bian et al., 2011
Cathepsin L (IrCL1)
35kDa
Ixodes ricinus Not tested Franta et al., 2011
P- selectin-binding protein
(Om44), 44kDa
Ornithodorous moubata Pigs Reduction in fecundity- 44%; feeding inhibition 50%
Garcia-Varas et al., 2010
Calreticulins
55-60kDa
Haemaphysalis longicornis Mice and calves Immunogenicity tested and proposed as good target
Parizi et al., 2009
RH50; 50kDa Rhipicephalus haemaphysaloides Rabbit Mortality rate 30.5% Zhou et al., 2006b
Ferritin 2 R. microplus calves E% = 64 Hijdusek et al., 2010
64TRP R. appendiculatus Rabbit E%- 50-70 Trimnell et al., 2002; 2005
Subolesin R. microplus calves E%- 50-75 Almazan et al., 2010
Voraxin-alpha R. appendiculatus Rabbit E%- 40-50 Yamada et al., 2009
Ubiquitin R. microplus Calves E%- 30-50 Almazan et al., 2010
Subolesin
It is ortholog of akirin, an evolutionary conserved gene of insect and
vertebrate (Mangold et al., 2009)
First discovered in Ixodes scapularis by Almazan et al., 2003
The proposed function of akirins is as transcription factors required for
NF-kB-dependent gene expression (Galindo et al., 2009) and in regulation
of innate immune response in fruit fly (Goto et al., 2008)
Subolesin functions in ticks are the same as akirin in fruit fly (Goto et
al., 2008; Galindo et al., 2009; Zivkovic et al., 2010; de la Fuente et al.,
2008; 2010)
Immunization with recombinant Subolesin
Tick species Vaccine efficay (against adults)
Reference
Ixodes scapularis 71% Canales et al., 2009
Amblyomma americanum
66% de la Fuente et al., 2010
R. (B.) microplus 51% Almazan et al., 2010
R. (B.) annulatus 60% Almazan et al., 2010
Vaccination with Subolesin reduced the vactor capacity of Ixodes scapularis for
Anaplasma phagocytophilum (Almazan et al., 2010; de al Fuente et al., 2010)
Merino et al. (2011) --- 98% and 99% reduction in infection level of A.
marginale and Babesia bigemina, respectively in R. (B.) microplus fed on
Subolesin immunized animal.
Calreticulins (CRT)In general, CRT is a calcium binding protein
Found in almost every organism
In ticks, the salivary secreted CRT involvement in evading the host's
immune system (Xu et al., 2005)
Kaewhom et al. (2008) reported, CRT is a protein found in tick
salivary glands and saliva, and CRT might facilitate tick feeding and
pathogen transmission through anti-thrombotic and complement-
inhibition activities.
Vaccination of sheep with rHqCRT conferred protective immunity
against Haemaphysalis qinghaiensis, resulting in 54.3% mortality in
adult ticks, compared to the 38.7% death rate in the control group (Gao
et al., 2008)
The possibility of using CRT to induce protective immunity against
Necator americanus and Schistosoma spp. has been suggested (El
Gengehi et al. 2000; Khalife et al. 1994; Pritchard et al. 1999).
Exhibition of necrotic lesions in the tick bite sites in Amblyomma
americanum CRT immunized rabbits indicates that immune reaction
could disrupt the feeding cycle (Jaworski et al. 1995).
Sanders et al. (1999) reported the antibody levels to A. americanum
CRT increase in humans after exposure to I. scapularis are
correlated with tick engorgement indices
Cathepsin LCathepsin family having dozen of member with protease activity.
Cathepsin L is a Cysteine protease
Sojka et al. (2008) and Horn et al. (2009) demonstrated that intestinal
haemoglobinolysis in the Ixodes scapularis relies on Clan CA papain-type
cysteine peptidases, cathepsins L (IrCL), B (IrCB) and C (IrCC), the Clan
CD asparaginyl endopeptidase, legumain (IrAE) and the Clan AA aspartic
peptidase, cathepsin D (IrCD)
Detailed analysis of the haemoglobinolytic pathway in the I. ricinus gut
demonstrated that the process is initiated by cleavage of large fragments
from haemoglobin by cathepsins D, L and Legumain
Franta et al., 2011
Silencing of IrCL by RNAi impaired weight-gain of semi-engorged
Ixodes ricinus females fed for 6 days on guinea pigs
This result suggests that IrCL has a non-redundant role in the
digestive machinery Franta et al., 2011
Targeting this enzyme using specific immunotherapeutic antibodies
provides a promising concept for the rational development of an
anti-tick vaccine (Jongejan et al., 2007)
Clara et al. (2011) through peptide phage display library shows
Cathepsin L is a potent digestive enzymes
Characterization of genes by gene silencing
RNA interference (RNAi) is a process within living cells that moderates the activity of
their genes.
Andrew Fire and Craig C. Mello, (1998) work on RNA interference in the C. elegans,
Nobel Prize in Physiology or Medicine 2006
RNAi has been shown to be valuable tools for the study of tick gene function, the
characterization of tick pathogen interface and the screening and characterization of
tick protective antigens.
de la Fuente et al., 2007
Nucleus
Cytoplasm
Exogenous dsRNA
ncRNAstRNA
mRNA
shRNA
siRNA
miRNA
Long ncRNA
Dicer
RISC
RISC
RISC
RNA gene
Tick species Target gene Phenotype References
A.americanum Histamine-binding protein (HBP)
Reduction of histamine-binding activity in salivary glands and aberrant tick feeding pattern
Aljamali et al., 2002; 2003
A.americanum Salivary Cystatin ~80% decrease in transcript level, 32% reduction in body weight, only 20% ticks was able to feed on host after injection
Karim et al., 2005
H. longicornis Leucine aminopeptidase
Delay onset of egg-laying and reduced oviposition
Hatta et al., 2007
Tick species Target gene Phenotype ReferencesR. (B.) microplus Subolesin reduction of 75%
and 99% in tick weight and egg mass, respectively and 46% mortality compare to control
Nijhof et al., 2007;
De la Fuente et al., 2005
H. longicornis Ribosomal protein P0 Low body weight, lower rate of engorgement, high mortality
Gong et al., 2008
R. (B.) microplus Ferritin 2 42% rejection, 50% reduction in weight, 53% reduction in oviposition
Hajdusek et al., 2009
Tick species Target gene Phenotype References
Amblyomma americanum
CD147 receptor homologue
~69% ticks are not able to feed properly, tick morphology was changed
Mulenga and Khumthong, 2010a
A. americanum Insulin like growth factor
Reduction in blood meal size, tick mortality, fail to lay eggs.
Mulenga and Khumthong, 2010b
R. (B.) microplus Metzincin metalloproteases
Affects average egg weight and oviposition rates
Barnard et al., 2011
R. (B.) microplus Ubiquitin-63E Knockdown of gene associated with Ubiquitin-63E
Lew-Tabor et al., 2011
Technical Program
A. Cloning and sequencing of targeted genes (Subolesin, Calreticulin and Cathepsin L) of Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus
B. Study on conservation of target genes among different isolates of H. a. anatolicum and R. (B.) microplus from India.
Ticks will be collected from different states (as much as possible).
Some isolates are already available in the Entomology laboratory,
Division of Parasitology, IVRI, Izatnagar and more will be
collected.
Total RNA will be isolated, target genes will be amplified using
suitable primers, cloned and sequenced.
Analysis of genes using bioinformatics software like Gene tool,
DNA star, Megaline, NCBI blast etc.
C. Quantification of level of transcript of targeted genes in different stages of H. a. anatolicum (IVRI line II)
Different life stages of ticks will be collected and kept in
RNAlater at -80°C
Total RNA will be isolated using standard protocol
Custom synthesis of primers
Quantification of transcriptome for each gene through
Quantitative PCR
D. Charecterization of targeted genes of H. a. anatolicum
1. Through gene silencing
2. In-vivo immunization study of recombinant
protein(s)
1. Gene silencing by RNA interference
I.Preparation of dsRNA (200-500bp) using standard protocolII.Inoculation of dsRNA in unfed adults of H. a. anatolicum
Dilution of dsRNA with injection buffer/elution buffer to make the concentration
@ 5.0 x 1010 to 5 x 1015 molecules/µl for each gene of interest.
1µl dsRNA preparations will be injected to the individual tick using specially
fabricated 34G needle fitted in micro-syringe (Hamilton, Switzerland) at posterior
to 4th coxae deep in to hemocele.
Injected ticks will be allowed to move in broad bottom tubes, incubate in BOD
incubator at 95% RH and 28°C temperature for 24 hours.
III. Assessment of biological activity of ticks
Active ticks will be selected (n = 30 for each gene inoculated and control) and
will be released on animal along with equal number of male ticks.
Feeding ticks (n= 10) will be collected at 24 hrs interval till engorgement and
will be stored in RNAlater at -80°C for RNA isolation. Engorged ticks will be
weighed and kept for oviposition at 28°C with 85% RH.
IV. Evaluation of effect of RNAi on ticks
Entomological parameters viz., percent reduction in tick number (DT%), percent
reduction in egg mass (DO%), percent reduction in tick weight (DR%) and overall
efficacy (E%) will be recorded and will be compared to control
Monitoring of inhibition of expression of gene(s) of interest in feeding ticks,
engorged ticks, eggs and larvae by q- PCR.
2. In-vivo immunization of recombinant protein(s)
I. Expression of target genes of H. a. anatolicum in prokaryotic system
Targeted genes will be expressed in suitable expression vector and
standardization will be done for good expression.
Purification and quantification of expressed protein (s).
Determination of molecular weight of recombinant proteins using
SDS-PAGE
Western blot analysis of recombinant proteins by probing with hyper
immune sera raised in rabbit against antigens prepared from H. a.
anatolicum
II. Immunization of calves with recombinant protein along with adjuvant
Cross bred caves of 3-4 month age from dairy farm (LPM), IVRI, Izatnagar
will be procured.
All the calves will be kept in tick proof shed of the Division of Parasitology.
All the animals will be dewormed after 15 days of arrivals.
Animals will be randomly divided in to different groups of four animals in
each group and immunization will be started on 6-7 month old calves.
Each animal of immunized group (s) will be inoculated with 100µg
of antigen along with adjuvant (1:1 ratio) in a three doses at one
month interval, deep intramuscularly
Control animals will be inoculated with PBS/adjuvant
For each antigen two groups will be kept, one will be challenged with
larvae (hatched from 50 mg eggs) and 50 unfed adults of H. a.
anatolicum and other with larvae (hatched from 50 mg of eggs) of R.
(B.) microplus
44
III. Monitoring of immunological response against immunogen
Serum will be collected at different time (pre-immunization, post-
immunization) for estimation of
Whole serum immunoglobulins
IgG1
IgG2
by indirect ELISA
IV. Potency testing by Entomological data
For the larvae-
DT (%) = 100 (1 – NTV/NTC)
where DT(%) is the percentage reduction of challenged larvae,
MO (%) = 100 (1-MLI/MLC)
where MO (%) is the percent reduction in moulting of engorged larvae,
For the adults- DT% = 100 (1-NTV/NTC),
Where DT% is the percentage reduction in mean number of females
fed on immunized and control groups of animals.
DO (%)= 100 (1- PATV/PATC)
where DO (%) is the percentage reduction of mean weight of eggs of
ticks fed on immunized and control animals
DR (%) = 100 (1- PMTV/PMTC)
where DR (%) is the percentage reduction of mean weight of adult
females dropped from immunized and control animals
E (%) = 100 [1- (CRT X CRO)]
Where E (%) is the efficacy of immunogens. CRO is reduction in egg
laying capacity (PATV/PATC), CRT is the reduction in the number of
adult females (NTV/NTC)
47