Outline Of Research Work Seminar Cloning and silencing of Subolesin, Cathepsin L andCalreticulin genes of Hyalomma anatolicum anatolicum and evaluation of cross-protective efficacy of recombinant protein(s) Dr. Binod Kumar Roll No.1374 Ph.D. Scholar Division Of Parasitology IVRI 1
TICKSMajor tick species in INDIA infesting livestockHyalomma anatolicum anatolicumCattle, Buffalo and small ruminantsThree hosts tickRhipicephalus (Boophilus) microplusCattle, Buffalo, Horse, donkeys, goat, Sheep, Deer, Pig, Dog, and somewild animalsOne host tick 4
Direct and Indirect effects Tick attachment Deep bite wound Annoyance Predispose to myasis Reduced hide value Tick secretions Tick toxicosis Tick paralysis Transmission of tick borne pathogens Blood feeding Anemia Production and reproduction losses Control High cost of acaricide treatment Ecological damage Human health
Vectorial capacityH. a. anatolicum•Theileria annulata•T. buffeli•T. lestocardi•CCHF (????)R. (B.) microplus Babesia bigemina B. bovis Anaplasma marginale 6
Economic impact of tick infestation onlivestock industry In million US $ De Castro, 1997; Minjauw and Mc Leod, 2003; Dayton et al., 1991; Horn, 1987; Mukhebi et al., 1999 * * Control cost 7
Ticks control strategiesMethods of Control Major Control Side effects of chemical Method control method Selection of resistant• Chemical control (Acaricides) ticks• Biological control Environmental pollution• Genetic transformation Acaricides Residues in livestock• Immunological control products• Herbal formulation as High cost of repeated acaricide application• Genetically resistance breeds Development of new generation acaricide Sustainable control ???????? 8
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 andefficacy 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 inHyalomma 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
Major areas of target 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
Targets selected for study Subolesin (Expressed in all organs and tissues) Function as transcription factors in theCalreticulin regulation of geneAnti-thrombotic and Cathepsin L expression Part of a gut-associated multi-complement-inhibition peptidase complex.activities in host Its endopeptidase activity is important in the initial phase of haemoglobinolysis.
•.. Objectives • Cloning and sequencing of Subolesin, Calreticulin and Cathepsin L genes of Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus • Evaluation of Conservation of target genes in different isolates of H. a. anatolicum and R. (B.) microplus • Characterization of target genes of H. a. anatolicum i). Through RNA interference ii). In-vivo immunization trial using recombinant protein(s)
Native proteins as Vaccine targetsImmunogen Challenge dose Percentage protection Larvae Nymphs Adults Immediate rejection (%)a Overall decrease in Reduction in successive stageb egg massesc AFF-TLE (L), 39kDa 4000 200 - 60.0 (L),44.0 (N) 34.0 (N),43.2 (A) Aff-GHLAg (Gut), 34 2000 140 40 pairs 24.2 (L),22.4 (N),32.2 (A) 31.2 (N), 25.2 (A), 15.0 kDa) Aff-HNAg (Nymph, 1600 140 40 pairs 38.0 (L), 25.0 (N), 32.2 (A) 32.7 (N), 28.7 (A), 20.0 39kDaAff-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) - a Mean differences in immediate rejection between immunized and control groups of animal. b Mean differences in the development of successive stage of the ticks fed on immunized and control group of animals. c Mean differences in the egg masses laid by the ticks fed on immunized and control group of animals. * p < 0.01. 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; ** p < 0.05 Indian J Anim. Sci., Exp. Appl. Acarol., 2003, Parasitology Res., 2005, Trop Anim Hlth Prod., 2005; J Vet Parasitol., 2007; Vaccine 2008
Recombinant proteins as Vaccine -Bm86 and its homologTickGARD™, Gavac™20-30% reduction in engorge tick number30% reduction in engorge tick weight60-80% reduction in egg masses50-60% reduction in number of acaricide treatment in a year (Willadsen et al., 1995, de la Fuente et al., 2007) 16
Variable efficacy of Bm86 based vaccine againstdifferent strain of R. (B.) microplus 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 0de la Fuente et al., 1995, 2005, 2006; Garcia-Garcia et al., 2000The use of Bm86 based vaccine on cattle tick strains located in differentgeographic areas has presented variable efficacy, even the so-called vaccinefailures that seemed to be due to variations in amino acids in the protein codifiedby 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)Results1% nucleotide variation within strains7.5% nucleotide variation between strainsThese variation cause change in amino acid composition at four placesSossai 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 geneGene 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 sufficientto confer vaccination inefficiencies when recombinant antigens are used.
Cross-protectionVaccine Tick species Effect ReferencemoleculesBm86 Hyalomma dromedarii DT%-27; DR%- 31; DO%- Rodríguez-Valle et 32 al., 2012Bm86 Amblyomma cajennense No effect Rodríguez-Valle et al., 2012Bm86 R. (B.) annulatus E%-99% Canales et al., 2009Bm86 R. (B.) decoloratus DT%- 45; DR%- 55; DO%- Odongo et al., 2007 61Bm86 Rhipicephalus sanguineus Reduction in Larvae, Perez-Perez et al., Nymph and Adults, 38%, 2010 29% and 31%, respectivelyBm86 R. appendiculatus No effect de Vos et al., 2001Bm86 Hyalomma anatolicum E%- 25 Kumar et al., 2012 anatolicum
Bm86 HomologRee86, Dr86, Hm86, Av86, Ir86, Os86 (Nijhof et al., 2010), Hl86 (Liao etal., 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 variableAntigens Tick species Efficacy ReferenceBa86 R. (B.) annulatus 83% Canales et al., 2009Ba86 R. (B.) microplus 71% Canales et al., 2009Haa86 H. a. anatolicum Larvae – 47- Azhahianambi et al., 60% 2009; Jeyabal et al., Adults - 40-80% 2010; Kumar et al., 2012Haa86 R. (B.) microplus 25% Kumar et al., 2012
Some other recently identified Vaccine targets-Targeted molecules Species of Ticks Experimen Vaccine Efficacy Reference tal animalAcid phosphatase (HL-3) Haemaphysalis longicornis Rabbit DR% = 10.6 Zhang et al., 2011 Mortality (%) = 28.041.0 kDaHc-23 Haemaphysalis concinna Rabbit DR% = 11 Bian et al., 2011 DO% = 6243 kDaCathepsin L (IrCL1) Ixodes ricinus Not tested Franta et al., 201135kDaP- selectin-binding Ornithodorous moubata Pigs Reduction in fecundity- Garcia-Varas et al., 44%; feeding inhibition 2010protein 50%(Om44), 44kDaCalreticulins Haemaphysalis longicornis Mice and calves Immunogenicity tested Parizi et al., 2009 and proposed as good55-60kDa targetRH50; 50kDa Rhipicephalus haemaphysaloides Rabbit Mortality rate 30.5% Zhou et al., 2006bFerritin 2 R. microplus calves E% = 64 Hijdusek et al., 201064TRP R. appendiculatus Rabbit E%- 50-70 Trimnell et al., 2002; 2005Subolesin R. microplus calves E%- 50-75 Almazan et al., 2010Voraxin-alpha R. appendiculatus Rabbit E%- 40-50 Yamada et al., 2009Ubiquitin R. microplus Calves E%- 30-50 Almazan et al., 2010
Subolesin•It is ortholog of akirin, an evolutionary conserved gene of insect andvertebrate (Mangold et al., 2009)•First discovered in Ixodes scapularis by Almazan et al., 2003•The proposed function of akirins is as transcription factors required forNF-kB-dependent gene expression (Galindo et al., 2009) and in regulationof 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 Reference (against adults) Ixodes scapularis 71% Canales et al., 2009 Amblyomma 66% de la Fuente et al., americanum 2010 R. (B.) microplus 51% Almazan et al., 2010 R. (B.) annulatus 60% Almazan et al., 2010Vaccination with Subolesin reduced the vactor capacity of Ixodes scapularis forAnaplasma 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 onSubolesin 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 hostsimmune system (Xu et al., 2005) Kaewhom et al. (2008) reported, CRT is a protein found in ticksalivary glands and saliva, and CRT might facilitate tick feeding andpathogen transmission through anti-thrombotic and complement-inhibition activities. Vaccination of sheep with rHqCRT conferred protective immunityagainst Haemaphysalis qinghaiensis, resulting in 54.3% mortality inadult ticks, compared to the 38.7% death rate in the control group (Gaoet 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 L Cathepsin 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 intestinalhaemoglobinolysis in the Ixodes scapularis relies on Clan CA papain-typecysteine peptidases, cathepsins L (IrCL), B (IrCB) and C (IrCC), the ClanCD asparaginyl endopeptidase, legumain (IrAE) and the Clan AA asparticpeptidase, cathepsin D (IrCD)•Detailed analysis of the haemoglobinolytic pathway in the I. ricinus gutdemonstrated that the process is initiated by cleavage of large fragmentsfrom 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 silencingRNA interference (RNAi) is a process within living cells that moderates the activity oftheir genes.Andrew Fire and Craig C. Mello, (1998) work on RNA interference in the C. elegans,Nobel Prize in Physiology or Medicine 2006RNAi has been shown to be valuable tools for the study of tick gene function, thecharacterization of tick pathogen interface and the screening and characterization oftick protective antigens. de la Fuente et al., 2007
Nucleus mRNA RNA gene tRNA s NA ncR miRNA shRNA Cytoplasm siRNAExogenous dsRNA Long ncRNA Dicer RISC RISC RISC
Tick species Target gene Phenotype ReferencesA.americanum Histamine-binding Reduction of Aljamali et al., 2002; protein (HBP) histamine-binding 2003 activity in salivary glands and aberrant tick feeding patternA.americanum Salivary Cystatin ~80% decrease in Karim et al., 2005 transcript level, 32% reduction in body weight, only 20% ticks was able to feed on host after injectionH. longicornis Leucine Delay onset of egg- Hatta et al., 2007 aminopeptidase laying and reduced oviposition
Tick species Target gene Phenotype ReferencesR. (B.) microplus Subolesin reduction of 75% Nijhof et al., 2007; and 99% in tick De la Fuente et al., weight and egg 2005 mass, respectively and 46% mortality compare to controlH. longicornis Ribosomal protein P0 Low body weight, Gong et al., 2008 lower rate of engorgement, high mortalityR. (B.) microplus Ferritin 2 42% rejection, 50% Hajdusek et al., reduction in weight, 2009 53% reduction in oviposition
Tick species Target gene Phenotype ReferencesAmblyomma CD147 receptor ~69% ticks are not Mulenga andamericanum homologue able to feed Khumthong, 2010a properly, tick morphology was changedA. americanum Insulin like growth Reduction in blood Mulenga and factor meal size, tick Khumthong, 2010b mortality, fail to lay eggs.R. (B.) microplus Metzincin Affects average egg Barnard et al., 2011 metalloproteases weight and oviposition ratesR. (B.) microplus Ubiquitin-63E Knockdown of gene Lew-Tabor et al., associated with 2011 Ubiquitin-63E
Technical Program• 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. anatolicum3. Through gene silencing4. In-vivo immunization study of recombinant protein(s)
1. Gene silencing by RNA interferenceIII.Preparation of dsRNA (200-500bp) using standard protocolIV.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 speciallyfabricated 34G needle fitted in micro-syringe (Hamilton, Switzerland) at posteriorto 4th coxae deep in to hemocele.•Injected ticks will be allowed to move in broad bottom tubes, incubate in BODincubator 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) andwill 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 willbe stored in RNAlater at -80°C for RNA isolation. Engorged ticks will beweighed 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%), percentreduction in egg mass (DO%), percent reduction in tick weight (DR%) and overallefficacy (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
III. Monitoring of immunological response against immunogenSerum will be collected at different time (pre-immunization, post-immunization) for estimation of Whole serum immunoglobulins IgG1 IgG2 by indirect ELISA 44
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 femalesfed on immunized and control groups of animals.DO (%)= 100 (1- PATV/PATC)where DO (%) is the percentage reduction of mean weight of eggs ofticks fed on immunized and control animalsDR (%) = 100 (1- PMTV/PMTC)where DR (%) is the percentage reduction of mean weight of adultfemales dropped from immunized and control animalsE (%) = 100 [1- (CRT X CRO)]Where E (%) is the efficacy of immunogens. CRO is reduction in egglaying capacity (PATV/PATC), CRT is the reduction in the number ofadult females (NTV/NTC)