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Vaccine 26S (2008) G40–G47
Contents lists available at ScienceDirect
Vaccine
journa l homepage: www.e lsev ier .com/ locate /vacc ine
rogress in development of vaccine against Hyalomma anatolicumnatolicum—Indian scenario
. Ghosha,∗, D.D. Raya, Vanlahmuakab, G. Dasc, N.K. Singhd,.K. Sharmaa, P. Azhahianambie
Entomology Laboratory, Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243122, Bareilly, Uttar Pradesh, IndiaDivision of Microbiology, Defence Research and Development Establishment, Jhansi Road, Gwalior, IndiaDepartment of Parasitology, College of Veterinary Sciences and Animal Husbandry, Rasalpura, Mhow 453446, Madhya Pradesh, IndiaDepartment of Veterinary Parasitology, GADVASU, Punjab, IndiaCentre for Biosystems Research, University of Maryland Biotechnology Institute, Rockville, MD, USA
r t i c l e i n f o
eywords:yalomma anatolicum anatolicumaccineontrol
a b s t r a c t
Hyalomma anatolicum anatolicum, a three host tick vector transmitting the causative agent of bovinetropical theileriosis, is widely distributed throughout India. As a component of integrated control measuresagainst the tick vector, attempts have been made to identify candidate protein molecules for developmentof an anti-tick vaccine in the different stages of this tick species. By strategic methods of isolation ofthe targeted molecules using affinity purification of proteins showing reactivity with immunoglobulins
of animals previously immunized with different sources of tick antigen, six proteins were isolated in asignificantly pure form. The recovery percentage of the candidate proteins was very low in the range of1.8–8.0%. The protective potentiality of the antigens was tested in immunization and challenge trials andmaximum potential was observed in the proteins isolated from total larval extracts, nymphal extracts andin larval glycoprotein. One of the antigens with a molecular weight of 37 kDa isolated from larvae of H. a.anatolicum was found to have some adverse effect on development of Theileria annulata in the vector tick.pme
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The progress in the develo
. Introduction
Ticks parasitize terrestrial vertebrates and transmit pathogenshat affect animal and human populations [1,2]. Tick infestationsmpact cattle production worldwide, for example infestations withhe cattle tick Boophilus microplus economically impact cattle pro-uction by reducing weight gain and milk production, and also byransmitting pathogens [3]. In India, livestock production is pri-
arily undertaken by small holding farmers who integrate croproduction with livestock keeping. Currently, livestock productionepresents one of the most promising fields of diversification forhe economy. Its products are a most important source of animalrotein and account for over 25% of the agricultural gross domes-ic product (GDP) [4]. However, productivity of these animals is
onstrained by direct and indirect effects of ticks. As per the recentstimate the annual cost of controlling ticks and tick borne diseasesf Indian animals is within 498.7 million US$ [5].∗ Corresponding author. Tel.: +91 581 2302368; fax: +91 581 2302368.E-mail address: [email protected] (S. Ghosh).
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264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2008.09.067
nt of immunoprophylactic measures against H. a. anatolicum is discussed.© 2008 Elsevier Ltd. All rights reserved.
Control of tick infestation has been difficult because ticks haveew natural enemies. The use of chemical acaricides is at presenthe major tick control strategy that is widely available. However,se of acaricides has had limited efficacy in reducing tick infesta-ions and is often accompanied by serious drawbacks, including theelection of acaricide resistant ticks, environmental contaminationnd contamination of milk and meat products with drug residues6]. Other approaches include use of hosts with natural resistanceo ticks, use of biological control agents and tick vaccines [7,8]. Theeasibility to control tick infestations through immunization haseen well documented and the technology has been adapted in aumber of countries [9].
Hyalomma anatolicum anatolicum, the vector of Theileria annu-ata, is widely distributed throughout India [10]. Control of theick infestation has been prioritized to reduce the direct effect onhe host and also to control the fatal parasite. Due to large scalend continuous use of acaricides for the control of the tick vector,
esistance has been reported from throughout the country [11]. Tovercome the problem, development of immunoprophylactic mea-ures against the vector tick has been given priority. The presentaper deals with the updated development in the search of formu-ating a vaccine against H. a. anatolicum.
ine 26
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. Material and methods
.1. Animals
Healthy cross-bred (Bos taurus × Bos indicus) male calves wererocured from the institute’s dairy farm and were housed on slat-ed floor in tick proof pens, provided with milk, calf-starter rationnd green fodder. The animals as well as the shed were kept tickree throughout the period of the experiments. The tick naive statusf these calves was confirmed by enzyme linked immunosorbentssay (ELISA) with antigens prepared from larvae, nymphs anddults of H. a. anatolicum. For all the experiments, at the time ofmmunization the calves were 8–12 months of age. They were ran-omly divided into immunized and control groups.
.2. Rearing of ticks
H. a. anatolicum were maintained at the Entomology laboratoryf the Division of Parasitology of the institute as per the method ofhosh and Azhahianambi [12]. For feeding of larvae and nymphsf H. a. anatolicum New Zealand white rabbits were used whileisease-free crossbred calves were used for feeding of adults. Larvaef B. microplus were fed on disease-free calves.
.3. Preparation of antigens
.3.1. Larval antigens of H. a. anatolicum (TLEH)Laboratory-reared, clean 5–6 days old unfed larvae were homog-
nized in cold phosphate buffered saline (PBS: 0.15 mM PBS, pH.2 with 1 mM disodium EDTA, pH 7.2) containing cocktail pro-ease inhibitors (1 mM ethylenediamine tetraacetic acid, ethylenelycol bis N,N,N,N-tetraacetic acid, N-ethylmaleimide and phenyl-ethylsulphonyl fluoride), filtered, sonicated and centrifuged at
5,000 × g for 60 min at 4 ◦C. The supernatants were designated asarval antigens of the respective species. The protein concentrationf the antigen was determined according to Lowry et al. [13].
.3.2. Nymphal antigen of H. a. anatolicum (HNAg)Unfed nymphs were surface sterilized in 3% H2O2, 70% alco-
ol and in PBS. Thereafter, the surface sterilized nymphs wereriturated in sterile glass mortar with PBS containing 1 mM dis-dium EDTA, 0.02% merthiolate, 0.01% PMSF and 0.075 mM sodiumeoxycholate. The homogenate was sonicated and centrifuged asbove and the supernatant was collected. The pellets were againuspended in extraction buffer and centrifuged for 1 h at 4 ◦C.upernatants were pooled, dialysed against 0.15 mM PBS and 1 mMisodium EDTA, pH 7.2. The detergent free extract was designated
s HNAg and was used for purification..3.3. Adult antigen of H. a. anatolicum (HAAg)Partially fed (4–5 days) adult female ticks were surface sterilized
s above and used for preparation of whole adult extracts using
iwamS
able 1etails of ligands used in the purification of tick antigen, molecular weight of the antigen
ntigen source Ligand used
arvae IgG from calves immunized with soluble larval antigen of H. a.anatolicum and protected against challenge infestations
arvae Anti-H. a. anatolicum gut IgGymphs IgG from rabbits immunized with soluble nymphal antigen and
protected against challengearvae Anti-gut IgG and concanavalin Adults Anti-gut IgGarvae IgG from animals immunized with gut origin larval antigen
and 60–70% protected against larvae and nymphal challenge
S (2008) G40–G47 G41
xtraction buffer. Method of preparation of antigen was same asentioned in the preparation of nymphal antigen.
.3.4. Gut antigen of H. a. anatolicum (HGAg)Mid gut was dissected from partially fed adult female ticks,
omogenized in 0.1 M PBS (pH 7.2) containing 1 mM mixture ofrotease inhibitors and 0.02% merthiolate. The homogenate wasonicated under cooling at 8 �m amplitude four times of 30 s eachith interspersed breaks of 10 s, centrifuged at 14,000 × g at 4 ◦C for
0 min and the supernatant was collected. The pellet was again dis-olved in extraction buffer, sonicated, centrifuged and supernatantas collected, pooled, filtered through 0.45 �m filter (Sartorius)
nd used as gut antigen (HGAg).
.4. Raising of anti-HGAg antibodies
For raising anti-HGAg antibodies three injections were given towo rabbits at days 0, 7 and 21. The first two injections were givenubcutaneously with 700 �g of HGAg in 500 �l PBS emulsified withn equal volume of IFA per rabbit. The third one was given intra-uscularly with 550 �g HGAg in 393 �l PBS. Two rabbits were kept
s control and were injected simultaneously with equal volumes ofBS and IFA. All the rabbits were bled intracardially on days 17, 28nd 34 after the first injection and the sera were stored at −20 ◦C.ntibody titre was measured by ID [14] and ELISA [15].
.5. Purification of antigens
The antigens prepared from larvae, nymphs and adults weretrategically purified using different affinity ligands as outlinedn Table 1. Purification steps were as follows: for purification ofntigens (TLEH, HNAg) using IgG ligands immunoglobulins wererecipitated from the respective sera with 40% ammonimum sul-hate [16]. The IgG was further purified on a DEAE-Sepharoseolumn (Pharmacia Fine Chemicals, Sweden) that had previouslyeen equilibrated with equilibrating buffer [17]. The purified IgGas dialysed extensively for 36 h at 4 ◦C against 1 M NaHCO3, 0.5 MaCl, pH 8.4 and coupled to cyanogen bromide activated SepharoseB as recommended by Pharmacia Fine Chemicals. The excessiveeactive sites were blocked by 0.2 M glycine buffer, pH 8.0. Therepared gel was pre-eluted (0.1 M glycine–HCl, pH 2.8) and equili-rated with buffer containing 10 mM sodium phosphate and 5 mMisodium EDTA, pH 7.2 before use. The column was regeneratedy using 0.1 M Tris–HCl, 0.5 M NaCl, pH 8.5; 0.1 M sodium acetate,.5 M NaCl, pH 4.5 after each use. Antigens were loaded separately
n the respective columns in batches at a flow rate of 40 ml/h. Theound antigens were then eluted with 0.1 mM glycine–HCl, pH 2.8,
nto tris base to increase the pH to 7.3. The eluted bound antigensere dialysed extensively against phosphate buffer, concentrated
nd protein concentration was determined by a spectrophoto-etric method [18]. The concentrated proteins were resolved by
DS-PAGE [19] and used for immunization.
s and percentage recovery.
Molecular weight (kDa) Recovery (%) Reference
39 6.0 Ghosh et al. [56]
100, 59.4, 37 3.8 Das et al. [60]39 8.0 Sharma et al. [58]
34 2.2 Singh and Ghosh [57]106.7, 68 4.5 Das et al. [59]37 1.8 Das et al. [53]
G42 S. Ghosh et al. / Vaccine 26S (2008) G40–G47
Table 2Details of immunization schedule, dose, adjuvant used and route of delivery.
Immunogen Schedule of immunization (week) Adjuvant Total dose per animal Route Reference
Aff-TLE (experiment 1) 0, 2, 4 IFA 2 mg SC [56]Aff-GHLAg (experiment 2) 0, 2, 4 FCA/IFA 1.6 mg SC/IM [60]Aff-HNAg antigen (experiment 3) 0, 2, 4 FCA/IFA 1.6 mg SC/IM [58]AHH
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LE: Total larval extract; GHLAg: gut origin larval antigen; HNAg: nymphal antigen;
For isolation of Aff-GHLAg and Aff-GHAAg, sera (titer 1:12,800)rom rabbits hyperimmunised with HGAg were used for isolationf IgG and the antigens were purified as mentioned above.
For isolation of larval glycoprotein (HGLA), Aff-GHLAg antigenas further purified as per the method of Snary and Hudson [20]
nd Parkhouse et al. [21]. Briefly, sterile 5 ml syringes were usedor setting up 2 ml immobilised ConA (Pharmacia Fine Chemicals,weden). The column was washed extensively with running buffer10 mM Tris–saline containing 1 mM CaCl2, 0.1 mM MnCl2 and 0.5%riton X-100). After washing, the column was preeluted with 0.1 M-methyl mannoside in running buffer followed by equilibrationith running buffer. The previously equilibrated Aff-GHLAg was
oaded on the column and bound glycoprotein was eluted. Theluted glycoprotein was immediately processed for equilibrationith phosphate buffer, pH 7.3 concentrated and designated asGLA. The antigen was resolved by SDS-PAGE on 1.0 mm thick gelssing a discontinuous system [19] and the glycoprotein nature ofhe isolated protein was confirmed by staining with periodic acidilver stain as per the method of Oakley et al. [22] with some mod-fications [23].
.6. Immunization and challenge schedule
For immunization experiments number 1–6, 8–12 months oldross-bred male calves were maintained in a tick proof animal shed.he tick free status of the animals were maintained by periodi-al treatment with insecticides on the animals and on the animalhed. The insecticide application was suspended 45 days before pri-
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able 3howing details of challenge infestation and post-challenge entomological parameters.
mmunogen Challenge dose Percentage protection
Larvae Nymphs Adults Immediate rejection (%)a
FF-TLE (experiment 1)d 4000 200 – 60.0 (larvae)*
44.0 (nymphs)*
ff-GHLAg (experiment 2)2000 140 40 pairs 24.2 (larvae)**
22.4 (nymphs)**
12.5 (adults)
ff-HNAg (experiment3)1600 140 40 pairs 38.0 (larvae)*
25.0 (nymphs)32.2 (adults)*
ff-GHAAg (experiment 4) 2000 140 – 10.3 (larvae)
GLA (experiment 5)2000 – 25 pairs 39.0 (larvae)*
28.0 (adults)*
GLA (experiment 6)3000 – 75 pairs 32.0 (larvae)
23.4 (adults)
HLgP (experiment 7) – 140 – 17.0 (nymphs)
a Mean differences in immediate rejection between immunized and control groups of ab Mean differences in the development of successive stage of the ticks fed on immunizec Mean differences in the egg masses laid by the ticks fed on immunized and control grd Experiment number.* p < 0.01.
** p < 0.05.
FCA/IFA 1.7 mg SC [59]FCA/IFA 200 �g SC/IM [57]Saponin 200 �g IM [61]
g: adult antigen; HGLA: gut origin larval glycoprotein.
ary immunization. For all the experiments animals were dividedandomly and 5 animals each were kept in immunized and unim-unized control groups. The schedule, adjuvant used, dose and
oute of delivery are given in Table 2.To evaluate the protective potentiality of the isolated immuno-
ens animals were experimentally challenged by laboratory reared. annulata free larvae, nymphs and adults of H. a. anatolicum. Theetails of the challenge dose is given in Table 3. The entomologicalata were collected and analyzed as follows:
a. Recovery% = (number of fed larvae; nymphs; adults;dropped)/(number of larvae; nymphs; adults; released). Thepercent rejection was calculated as 100 − percentage recovery.
. Moulting% = number of subsequent stages emerging after moult-ing/total number of tick stages feeding to engorgement.
c. Egg mass (mg) = eggs laid by individual adult were weighed.. Reduction% = (mean weight of eggs laid by the ticks fed on immu-
nized animals)/(mean weight of eggs laid by the ticks fed oncontrol animals) × 100.
For experiment number 7, FCA/IFA was replaced with com-ercially and ethically acceptable adjuvant for the immunization
chedule and the temporal variation in entomological parameters
as studied by repeated challenge infestations, a condition thatimulates the tick infestations in the field. This immunization trialas conducted using saponin as an adjuvant in place of FCA/IFA
nd the immunoprotective potential of the 34 kDa glycoprotein wasested under repeated challenge conditions. Experimental calves
Reference
Overall decrease in successive stageb Reduction in egg massesc
34.0 (nymphs)* – [56]43.2 (adults)* –
31.2 (nymphs)* – [60]25.2 (adults)** –– 15.0
32.7 (nymphs)* – [58]28.7 (adults)* –– 20.0**
17.6 (nymphs) – [59]
16.0 (nymphs) – [57]– 15.8
29.32 (nymphs) – [61]– 50.67
20.7 (adults) – [53]
nimal.d and control group of animals.oup of animals.
S. Ghosh et al. / Vaccine 26S (2008) G40–G47 G43
Table 4Details of immunization/infection dose and delivery schedule.
Immunization/infection Time (day) Group Route
I II III IV
Primary 0 400 �ga 400 �ga PBS PBS SCFirst booster 14 400 �ga 400 �ga PBS PBS SCInfection with T. annulata 21 0.5 tick – 0.5 tick – SCS
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ere divided into two groups and immunized with HGLA or adju-ant as control. HGLA was thoroughly mixed with saponin (1 mgaponin/ml of antigen) and injected to animals of group A. Theroup B animals were inoculated with an equal volume of PBS plusaponin. The details of the immunization schedule, dose, adjuvantsed and route of delivery are given in Table 2 (experiment 6). Allhe calves were experimentally challenged on week 5, 7, 9, 16 and9 after the first immunization (WFI) by adults and larvae of H. a.natolicum released on ears by the ear bag method. The challengechedule up to 25th WFI is elaborated in Table 3. The ear bags werehecked daily and post-challenge data were recorded.
In experiment number seven, cryopreserved stabilate of T. annu-ata (Parbhani) obtained from ground up tick supernatant (GUTS)
as used for experimental infection. Twelve bovine calves wereandomly distributed in 4 groups of 3 calves each. Calves of group Ind II were immunized with GHLAgP emulsified with an equal vol-me of Freund’s incomplete adjuvant (FIA). The calves of group I and
II were infected with 0.5 tick equivalent stabilated sporozoites of T.nnulata. The calves in group IV were kept as control. The schedule,ose and route of delivery are given in Table 4. Each of the calvesf groups I–IV were challenged (Table 3) and the post-challengearameters were recorded and analyzed statistically [24].
.7. Assessment of T. annulata in salivary glands of ticks
In experiment seven, the adults of H. a. anatolicum devel-ped from the nymphs used in challenge infestation on each ofhe calves of group I (immunized and infected) and group IIIinfected) were partially fed on rabbits for 72 h. Twenty partiallyed adult ticks, male and female in equal numbers, were randomlyelected from each calf. The salivary gland of the ticks were dis-ected and the left salivary gland of each tick was stained withethyl green pyronin (MGP) [25] and the degree of infectionith T. annulata was expressed as prevalence (number of infected
icks/number of ticks examined × 100), abundance (number of par-site masses/number of ticks examined) and intensity (number ofarasite masses/number of ticks infected) [26]. The right salivaryland of each tick was separately homogenized in 2 ml plastic vialsith 300 �l of Tris–HCl buffer (pH 8.0) containing 0.4 M NaCl andmM EDTA. Genomic DNA was extracted and PCR reaction was setp using 17mer primers encoding for the 30 kDa major merozoiteurface antigen of T. annulata [27]. The PCR cycle condition was 35ycles at 94 ◦C for 1 min, at 50 ◦C for 1 min and extension at 72 ◦Cor 1 min. The intensity of the PCR product bands were recorded byisual ranking as 0 (nothing seen), 1 (weak), 2 (moderate) and 3strong).
For assessment of sporozoite infectivity, GUTS were preparedrom 20 randomly selected partially fed adults of H. a. anatolicum
omprising male and female ticks in equal numbers emanatingrom each of the experimental calves of groups I and III. The GUTSere filtered and diluted with RPMI 1640 supplemented with 3.5%ovine serum albumin (BSA) to give a concentration of 4 ticks perl. This was serially diluted in a volume of 0.5 ml with RPMI/BSA ini1itf
200 �gb PBS PBS IM
4 well tissue culture plates. The mononuclear cell were separatedrom a normal bovine calf according to Boyum [28] and suspendedn RPMI 1640 supplemented with 20% foetal calf serum. The sus-ension of mononuclear cells in the volume of 0.5 ml (106 cells)as added to 0.5 ml of GUTS in each well of the tissue culture plate.
he plates were incubated at 37 ◦C in a 5% CO2 atmosphere. Titerf infectivity of sporozoite (highest sporozoite dilution infecting ateast 1% lymphocytes) and maximum percentage of infection wereetermined after 4 days of incubation.
.8. Dynamics of antibody response
Animals of all the experiments (1–7) were bled, sera were sep-rated and tested by ELISA [29]. All the reagents were optimizednd 96-well microtitre plates were coated with respective antigensiluted in carbonate bicarbonate buffer, pH 9.6. Sera from all thenimals collected on different days of immunization were seriallyiluted and tested in triplicate wells. The specific antibody responseas measured using secondary antibody (peroxidase conjugated
nti-bovine IgG (1:10,000), Sigma Chemical Company, USA). Finally,PD (orthophenylenediamine in phosphate citrate buffer, pH 5.0,igma Chemical Company) was used for colour development. TheD values were measured at 492 nm using BioRad ELISA reader. Forll the steps, incubation time was kept at 2 h except the last stagehen plates were kept for 20–30 min in the dark.
.9. Statistical analysis
Significant differences in mean values from immunized and con-rol animals were determined using student’s t-test [24].
. Results
.1. Purification of tick antigens
Table 1 shows the molecular weight of the proteins isolated fromifferent stages of the tick using different ligands. Amongst the sixroups of proteins isolated, highest recovery of 8% was recordedn the case of Aff-HNAg and the lowest was GHLAgP (1.8%). Of theifferent proteins isolated maximum purification was achieved inhe isolation of HGLA.
.2. Effect of immunization on tick stages
The details of the effect of immunization of animals on lar-ae, nymphs and adults of H. a. anatolicum has been summarizedn Table 3. Of the six antigens tested, highest percentages (60%)f challenged larvae were immediately rejected from the animals
mmunized with Aff-TLE (experiment 1) and lowest efficacy of0.3% was recorded in animals immunized with Aff-GHAAg (exper-ment 4). Besides the immediate effect of the tested antigens,he population control effect was also studied and Aff-TLE wasound highly efficacious in limiting the development of a significant
G ine 26S (2008) G40–G47
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Table 5Rate of infection with T. annulata in adults of H. a. anatolicum.
Group I Group III
Prevalence (%) 75.0 ± 0.00a 85.0 ± 2.887Intensity 2.38 ± 0.211 2.80 ± 0.226Abundance 1.78 ± 0.207 2.38 ± 0.232PCR (%) 83.33 ± 3.33 90.0 ± 2.88
Group I, Tick resistant calves; Group III, Tick naïve claves.a p < 0.01.
Fig. 1. Amplification of a 721 bp DNA fragment of T. annulata in salivary glands of(I1g
3Table 6 shows the in vitro infection pattern of bovine mononu-
clear cells by the GUTS. The GUTS of the ticks originating from thecalves of group III showed a higher titre of infectivity (10−14 to10−17) and resulted in higher infection in cells (5–8%) in comparison
44 S. Ghosh et al. / Vacc
ercentage of the nymphal population. Although the immediateejection percentages of larvae fed on Aff-GHLAg, Aff-HNAg andGLA immunized animals (experiments 2, 3 and 6) was 35.8%, 22.0nd 28%, respectively, less than the larvae fed on immunized ani-als of experiment 1, the overall decrease in the development of
uccessive stage was highly comparative.Amongst the six antigens, five antigens were tested against
ymphal challenge and animals immunized with Aff-TLE provedost efficacious in rejecting maximum percentages of challenged
ymphs from the immunized animals. Interestingly, although ani-als in experiment 3 were immunized with homologous antigen,
he protective efficacy was higher in Aff-TLE than Aff-HNAg. Sub-equent to feeding of ticks in animals of experiment 3, 28.7% moreymphs were unable to moult to the next stage in comparison tohe control while it was 43.2% in case of ticks fed on animals ofxperiment 1.
Of the seven experiments conducted, animals were challengedith adult ticks in experiments 2, 3, 5 and 6. It was interesting toote that although the immunizing dose was eight times less inxperiments 5 and 6 in comparison to the dosages given in exper-ments 2 and 3, the immediate rejection percentage of adults fedn the animals of experiment 5 was comparable to ticks fed onnimals of experiment 3 while the effect of Aff-GHLAg on adulticks was significantly lower than the other two antigens. Besideshe significant effect of the antigens on immediate rejection ofhallenged adults, the engorgement weight of the adults fed onnimals of experiments 3 and 5 was significantly reduced in com-arison to the control and subsequently, 15–20% reduction in eggasses was recorded. When challenge data of experiments 5 andwere compared, it was observed that animals immunized withGLA plus saponin conferred high level of protection in compari-
on to the ticks fed on animals immunized with HGLA plus FCA/IFA.he population control potentiality (50.67% reduction in eggass) of the adjuvant formulation (saponin in mineral oil + HGLA)as highest in comparison to any of the formulation tested
o far.It is interesting to note that a significant percentage of larvae
7–10%) fed on animals of experiments 1–3, 5 and 6 and nymphs4–9%) fed on animals of experiments 1–3 were abnormally fedharacterized by pale yellow in colour. Most of the abnormally fedarvae/nymphs could not moult to the next stage of development.
.3. Experiment 7: immunoprotective potential of the 34 kDalycoprotein tested under repeated challenge conditions
.3.1. T. annulata infection in calvesThe symptoms of mild infection of T. annulata like slight swelling
f the left pre-scapular lymph nodes and transient fever (rectalemperature above 39.5 ◦C) were recorded in all calves of groupand III on day 3 to day 8 post-infection. Biopsy smears of enlargedymph nodes showed the presence of 0.6–1.2% macroschizontsnfected lymphoblasts. The percentage of erythrocytic infectionas between 1 and 2% when the nymphs were fed on the calves.
.3.2. Infection in H. a anatolicumTable 5 summarizes the rate of infection with T. annulata in
dults of H. a anatolicum. In MGP staining, the prevalence, intensitynd abundance of infection in the left salivary gland was higher inhe ticks fed on the calves of group III than in the ticks fed on calves
f group I. PCR revealed higher prevalence of infection in ticks thated on calves of group III. In 60 ticks (male and female in equal num-ers) examined from each group, 83.4 ± 3.3% (n = 50) were positiven PCR in group I and 90.0 ± 2.8% (n = 54) were positive in group IIIFigs. 1 and 2).
F(I1g
A) female (n = 30) and (B) male (n = 30) H. a. anatolicum ticks fed on calves of group. Lane M, marker; lane P, control DNA; lane T, tick DNA; lane N, no template; lanes–10, DNA samples extracted from the salivary glands from ticks fed on animals ofroup I.
.3.3. In vitro infectivity of sporozoites
ig. 2. Amplification of a 721 bp DNA fragment of T. annulata in salivary glands ofA) female (n = 30) and (B) male (n = 30) H. a. anatolicum ticks fed on calves of groupII. Lane M, marker; lane P, control DNA; lane T, tick DNA; lane N, no template; lanes–10, DNA samples extracted from the salivary glands from ticks fed on animals ofroup III.
S. Ghosh et al. / Vaccine 26
Table 6In vitro infection pattern of bovine mononuclear cells by sporozoites of T. annulataharvested from adultsa of H. a. anatolicum.
Calf number Maximum infectedcells (%)
Titre of sporozoiteinfectivity
Group I 571 2.0 2−12
712 2.0 2−14
713 3.0 2−11
Group III 576 6.0 2−14
688 8.0 2−16
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roup I, Tick resistant calves; Group III, Tick naïve claves.a Batch of 10 male and 10 female ticks from nymphs fed on a single calf.
o the titre of infectivity (2−11 to 2−14) and maximum rate of infec-ion (2–3%) in cells recorded with the GUTS of the ticks originatingrom the calves of group I.
.3.4. Antibody responseA significantly high antibody response (p < 0.05) was recorded
n immunized animals (experiment 1) from 21st day post-mmunization (DPI) to 84th DPI and the high antibody responseas continued till the end of the experiment.
In a constant dilution of 1:1600, peak anti-aff GHLAg (experi-ent 2), anti-aff HNAg (experiment 3), anti- GHAAg (experiment 4)
nd anti-GHLgP (experiment 7) antibody response was noted on 35PI. Although a slight decrease in the antibody response was notedn 57th DPI, significantly high antibody response in comparison toontrol was recorded in all the immunized animals throughout theeriod of experiment.
At a dilution of 1:1000 peak anti-HGLA antibody response wasoted on 35th DPI and significant response in comparison to controlas noted up to 70th DPI. On the day of challenge, the immunized
nimals gave a titre of 1:8000 and was maintained up to 84th DPIexperiment 5). However, the antibody response in claves immu-ized with HGLA mixed with saponin (experiment 6) was increasedignificantly than the response noted in experiment 5. A peak anti-ody response (8.5× normal value) was recorded on 56th DPI andaintained up to 133th DPI. From 140th DPI although a decreasing
rend of antibody response was noted, the OD492 value of sera col-ected from the immunized animals remained high throughout theeriod of experiment (experiment 6).
. Discussion
In concert with the principles of sustainable agriculture,accines offer a number of advantages over conventional acari-ides/insecticides for the control of arthropod pests. The effectf immunisation can be long lasting and without the complica-ion of residues. Vaccines are environmentally safe and arthropodesistance to vaccines is less likely to occur [9,30]. It has beenemonstrated in IPM field trials that tick vaccine can be used toeduce the amount of insecticides load on the environment [31].
The idea of developing immunoprophylactic measures againstulti-tick infestations on crossbred animals was based on the con-
ept that ticks feeding on appropriately immunized hosts mightngest antibodies specific for (a) target antigen(s) within the tick,roducing a deleterious effect on their feeding and reproductiveerformances [32]. As one of the crucial steps in vaccine devel-
pment, concerted attempts were made to identify and isolateandidate vaccine molecules from the stages of targeted tick vec-ors. Following fractionation and testing methods a battery ofntigens have been isolated in a significantly purified form andested for their potentiality against homologous challenge infes-rrcw
S (2008) G40–G47 G45
ations. Earlier, different purification protocols have been used todentify and purify protective tick antigens [33–36]. In the case of. a. anatolicum, immunoaffinity chromatographic methods were
trategically employed for isolation of different protein molecules.lthough only in one case a very high level of purification waschieved (experiments 5 and 6), the targeted proteins were 80%f the eluent collected using elution buffer. One of the major issuesssociated with the isolation of the targeted protein is the lowecovery of the antigens. The recovery level of 1.8–6% achieved dur-ng the tedious purification protocol was only sufficient to test the
olecules in a limited number of experimental animals.Of the various entomological parameters used for evaluating the
uccess of immunization, a significant rejection of feeding ticks,eduction in development of successive generation and egg massesf adult ticks [37–39] are considered most important. Amongsthe six group of antigens tested the difference in mean immedi-te rejection of larvae from the immunized animals compared toontrol animals varied from 10.3 to 60% and highest efficacy wasecorded in experiment 1 where larval antigen was purified fromrude extract. In experiment 5, larval antigen was further purifiednd although the purification level was achieved at a very high levelhe immediate rejection level was reduced significantly in compar-son the data obtained with the crude extract. This may be due tohe additive effect of the other minor proteins present in the Aff-LE isolated by employing IgG ligand from the animals immunizedith crude larval extract and protected up to 60–70%. Comparing
he data generated in the seven experiments it can be concludedhat the antigens Aff-TLE, Aff-HNAg and HGLA are potent in limitinghe feeding process and development of H. a. anatolicum.
Of the different groups of adjuvants, the vesicle adjuvantCA/IFA, has been used extensively in combination with differentick derived antigens for immunization of rabbits and cattle againstxperimental challenge infestations [40–42]. However, FCA/IFAs not found suitable for food animals as it is highly toxic, canause chronic inflammation and induce autoimmune complica-ions [43,44]. In recent years therefore, a great deal of researchffort has been directed towards developing safe adjuvants whichave the potency but do not have the adverse effect of FCA/IFA.he surfactant adjuvant saponin, a complex mixture of triterpenoidxtracts from the bark of the Quillaia saponaria, has been identifieds safe, inexpensive and a potent adjuvant for veterinary vaccines.aponin induces strong Th1 and Th2 responses and moderate CTLesponses to proteins as a result of forming mixed protein–saponinicelles [45], and stimulation of Th1 and Th2 responses to tick
nfestations have been found to have significant impact in confer-ing protection against challenge infestations [46].
Earlier, in a comparative study, we evaluated the immunos-imulating properties of saponin and IFA in combination with anffinity purified 39 kDa antigen of H. a. anatolicum against experi-ental challenge infestations in rabbits [47]. Although insignificant
ifference in the antibody response of rabbits immunized withhe 39 kDa protein plus saponin/IFA was noted, a comparativelyigher level of protection against challenge tick stages was recorded
n the calves immunized by the saponin plus 39 kDa antigen. Inhe present investigation, a significantly high anti-HGLA antibodyesponse in comparison to the control was recorded in the calvesrom 5th to 17th weeks of immunization. It may be speculatedhat the generated antibodies conferred a significantly high level ofmmediate protection and interfered with the egg laying capacityf the feeding ticks.
An important impact of controlling tick infestations is theeduction of transmission of tick borne pathogens. Trials usingecombinant Bm86 antigen demonstrated vaccination against ticksontrolled the transmission of Anaplasma marginale in the regionshere ticks are the main vectors [48]. Recently, Labuda et al. [49]
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eported vaccination of mice with the putative tick cement protein4P prevented transmission of TBE virus. The association betweenhe tick vector and the pathogen was exemplified by Pal et al.50]. These authors have identified a tick receptor (TROSPA) thats required for Borrelia burgdorferi colonization of Ixodes scapularis.hey identified OspA as the bacterial ligand for TROSPA and demon-trated that the blockade of TROSPA by TROSPA antisera reduceddherence to the I. scapularis gut in vivo. In the present investi-ation there was an apparent reduction in the infection rate of T.nnulata in the salivary glands of adult ticks emanated from calvesf group I. This may be due to the damage of the gut of ticks fedn animals of group 1 immunized by GHLAgP. For survival and suc-essful transmission of T. annulata, there is a need for the zygote topecifically penetrate into viable gut digestive cells, then developo the kinete stage before migrating to the salivary gland. The effectf vaccination on gut digestive cells of B. microplus has previouslyeen reported [40] and the same mechanism may most likely beperating in the present vaccination trial leading to reduced pres-nce of T. annulata in the salivary glands of adult ticks emanatedrom the calves of group 1. The band intensities of the PCR productsere used as an indirect quantitative measurement of the number
f parasitized acini in the gland according to the method adoptedy Kirvar et al. [51]. A comparatively higher number of ticks devel-ped from calves of group I showed lower infection rates than theicks developed from calves from group III.
The in vitro infectivity test of GUTS indicated the presence ofore viable sporozoites in ticks fed on non-immunized calves
group III). Using a similar technique, Rubaire-Akiiki reported dele-erious effects of anti-tick immunity in cattle, produced by repeatednfestations with live nymphs of H. a. anatolicum on the viability of T.nnulata [52]. The observations in the present study suggest a par-ial inhibition in the growth rate of T. annulata due to immunizationith a purified larval antigen of H. a. anatolicum.
Although a reduction in the disease transmission potential oficks is achieved by vaccination against ticks [48,49,53], a cocktailaccine against both the tick and the pathogen/parasite is expectedo give an even higher level of protection against tick-borne dis-ases. A possibility for the development of such a combinationaccine against T. parva–R. appendiculatus and T. annulata–H. a.natolicum systems may exist. A novel subunit vaccine against T.arva has been recently evaluated for its vaccine potential in cat-le [54,55]. Similarly, in case of the T. annulata and H. a. anatolicumystem, three potential parasite targets and one vector target haveeen identified, genes have been cloned and recombinant proteinsave been generated in the Division of Parasitology, Indian Veteri-ary Research Institute. Research is underway to test the expressedntigens in different combinations to achieve the target of develop-ng integrated management strategy against the targeted tick andarasite species.
cknowledgements
Authors are grateful to Indian Council of Agricultural Researchnd Department of Biotechnology, Government of India for fund-ng. The preparation of the manuscript has been facilitated throughsian component of The Integrated Consortium on Ticks andick-borne Diseases (ICTTD-3), financed by the International Coop-ration Programme of the European Union through Coordinationction Project No. 510561.
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