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Veterinary Parasitology 188 (2012) 231– 238

Contents lists available at SciVerse ScienceDirect

Veterinary Parasitology

jo u rn al hom epa ge : www.elsev ier .com/ locate /vetpar

accine potential of recombinant antigens of Theileria annulata andyalomma anatolicum anatolicum against vector and parasite

. Jeyabala, Binod Kumara, Debdatta Raya, Palavesam Azahahianambib, Srikanta Ghosha,∗

Entomology Laboratory, Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243122, UP, IndiaCentre for Biosystem Research, University of Maryland Biotechnology Institute, Rockville, MD, USA

r t i c l e i n f o

rticle history:eceived 15 October 2011eceived in revised form 26 March 2012ccepted 28 March 2012

eywords:Haa86SPAG1TaSPyalomma anatolicum anatolicumheileria annulata

a b s t r a c t

In an attempt to develop vaccine against Hyalomma anatolicum anatolicum and Theileriaannulata, three antigens were expressed in prokaryotic expression system and protectivepotentiality of the antigens was evaluated in cross bred calves. Two groups (grs. 1 and 4)of male cross-bred (Bos indicus × Bos taurus) calves were immunized with rHaa86, a Bm86ortholog of H. a. anatolicum, while one group of calves (gr. 2) were immunized with cocktailsof two antigens viz., surface antigens of T. annulata (rSPAG1, rTaSP). One group each was keptas negative controls (grs. 3 and 5). The animals of groups 1, 2 and 3 were challenged withT. annulata infected H. a. anatolicum adults while the animals of groups 1, 3, 4 and 5 werechallenged with uninfected adult ticks. A significantly high (p < 0.05) antibody responses toall the three antigens were detected in immunized calves, but the immune response wascomparatively higher with rHaa86 followed by rTaSP and rSPAG1. Upon challenge with T.annulata infected ticks, animals of all groups showed symptoms of the disease but there

was 50% survival of calves of group 1 while all non immunized control calves (group 3)and rSPAG1 + rTaSP immunized calves died. The rHaa86 antigen was found efficacious toprotect calves against more than 71.4–75.5% of the challenge infestation. The experimenthas given a significant clue towards the development of rHaa86 based vaccine against bothH. a. anatolicum and T. annulata.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

Tick infestations significantly impact cattle productiony reducing weight gain, milk production and also byransmitting pathogens (Peter et al., 2004). In India, outf 106 reported tick species infesting animals, Hyalommanatolicum anatolicum, the vector of the apicomplexan par-site, Theileria annulata, is distributed widely (Ghosh et al.,007). Tropical theileriosis caused by T. annulata affect

attle in a large geographical areas of north Africa, south-rn Europe and most of the parts of Asia. In India, thisector–pathogen combinations is a significant contributor

∗ Corresponding author. Tel.: +91 581 2302368; fax: +91 941 0261029.E-mail address: sghoshp@yahoo.co.in (S. Ghosh).

304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.vetpar.2012.03.051

to control cost incurred in the tune of 498.7 million US$ perannum (Minjauw and McLeod, 2003).

Current control of H. a. anatolicum and T. annulatalargely focused on repeated use of acaricides whichhas limited efficacy and is often accompanied by seri-ous drawbacks viz., selection of acaricides-resistant ticks,environmental pollution and contamination of livestockproducts with acaricide residues (Graf et al., 2004). Othermethods of tick control include the use of hosts with nat-ural resistance to ticks, biological control and vaccines(Willadsen and Jongejan, 1999; de la Fuente and Kocan,2003; Willadsen, 2006). The feasibility of immunization of

hosts against cattle tick, Rhipicephalus (Boophilus) microplususing selected antigens (Bm86) was demonstrated and vac-cines have been developed for successful control of thetick species (de la Fuente and Kocan, 2006; Willadsen,

Parasit

232 L. Jeyabal et al. / Veterinary

2006; de la Fuente et al., 2007). The ortholog gene of Bm86was identified in other tick species and possibilities ofdevelopment of recombinant vaccines against these tickspecies have been explored (de la Fuente and Kocan, 2003;Azhahianambi et al., 2009a; Nijhof et al., 2010).

Immunological control of bovine tropical theileriosisis achieved by the use of attenuated schizont vaccine inIsrael (Pipano and Tsur, 1966), Iran (Hashemi-Fesharky,1988), Turkey (Sayin et al., 1997), Tunisia (Darghouth,2008), China (Zhang, 1997) and in India (Subramanianet al., 1988). However, the vaccine has limitations like therequirement of cold chain for preservation and the pos-sibility of reversion of the attenuated strain to virulentstrain. To overcome these problems, research for develop-ment of subunit vaccine against T. annulata infection hadbeen focused on surface antigens present on sporozoitesand merozoites (Boulter and Hall, 2000). Immunization ofanimals with different constructs of sporozoite surface pro-tein (SPAG1) provided partial protection in calves probablythrough reduction in the number of sporozoites in chal-lenge infection and their reduced ability to invade targethost cells (Boulter et al., 1998). Inclusion of more anti-gens of T. annulata, particularly from the schizont stage,had been emphasized to improve the level of protection(Preston et al., 1999).

Development of resistance in cattle against the vectorticks was shown to be effective in reducing the ability ofticks to transmit Theileria parva (Fivaz et al., 1989), T. annu-lata (Rubaire-Akiki, 1990; Das et al., 2005), Babesia species(de la Fuente et al., 2007) and tick borne encephalitis virus(Labuda et al., 2003). The ortholog of Bm86 was identi-fied in H. a. anatolicum having seven complete EGF-likedomain and the antigen was found protective in confer-ring protection against experimental challenge infestations(Azhahianambi et al., 2009a). In the present study, attemptwas made to evaluate the protective efficacy of rHaa86against the homologus challenge infestations and againstlethal dose of T. annulata. The result was compared with theefficacy of a cocktail of recombinant proteins of T. annulata(SPAG1 and TaSP) in conferring protection against theile-riosis in crossbred calves.

2. Materials and methods

2.1. Experimental animals

Thirty, one month old crossbred bovine male calves(Bos indicus × Bos taurus) were procured from institutedairy farm. The calves were housed individually in tickproof pens and maintained on milk, grain concentratesand wheat bhusa. The blood smears of the calves wereexamined for the presence of any hemoparasites and theirserum samples were examined for antibodies against sol-uble piroplasm antigen of T. annulata or larval antigen of H.a. anatolicum to ensure freedom from any previous expo-sures to vector and parasites. The experimental animals

were maintained as per the approved guidelines laid downby the committee for the purpose of control and supervi-sion of experimental animals (CPCSEA), a statutory Indianbody.

ology 188 (2012) 231– 238

2.2. Ticks

2.2.1. T. annulata free adult H. a. anatolicum ticksThe homogenous T. annulata free H. a. anatolicum, IVRI

line II was maintained in the Entomology laboratory ofthe Division of Parasitology as per standardized protocol(Ghosh and Azhahianambi, 2007). Healthy New Zealandwhite rabbits weighing 1.5–2 kg were used for feeding oflarvae of tick species. The adults were fed on disease freemale cross bred calves. The engorged adults were keptsingly in tick rearing glass vials maintained at 28 ◦C and85% RH in BOD incubator for egg laying.

2.2.2. T. annulata infected adults of H. a. anatolicumTwo four months old cross bred male calves were inoc-

ulated subcutaneously with a cryopreserved stabilate of T.annulata (Parbhani isolate) equivalent to 2 infected ticks.Uninfected nymphs were allowed to feed on the infectedcalves when intra-erythrocytic piroplasms were detectedin the blood smears (showing 30% of RBCs infected withpiroplasms of T. annulata). The fully engorged nymphs werecollected, reared in the laboratory. The molted adults werereared for seven to ten days before used for challengingimmunized and control calves.

2.3. Recombinant antigens

2.3.1. Mass culture of recombinant bacterial clonesrSPAG1: The details of expression of SPAG1 gene are

elaborated in Vanlalmuaka et al. (2010). The gene wascloned in expression vector pET32c(+) and transformedinto a BL21(DE3) pLysS strain of Escherichia coli. Expres-sion was induced with 1 mM IPTG and incubated at 37 ◦C inshaking incubator. The expression was confirmed by SDS-PAGE.

rTaSP: The details of expression of the gene are elabo-rated in Vanlalmuaka et al. (2010). In brief, the gene codingfor 26–156 amino acids of the TaSP gene was PCR amplifiedusing custom synthesized oligonucleotide primers: 5′F GTCGAC CAT GGA TCG ACA ACT TAA TCC 3′ and 5′R ATC TGCAGT ACC CGT CAG ACT CAT CAT C 3′. The amplified genewas cloned into pET32c(+) (Novagen, Merck Biosciences,Germany) for expression in BL21(DE3)pLysS E. coli. Expres-sion was induced with IPTG and expression profile wasanalyzed under denaturing condition.

rHaa86: The details of expression of the gene areelaborated in Azhahianambi et al. (2009a,b). On thebasis of published sequence information (AF347079), a1.755 kb size Bm86 ortholog gene (Haa86) was ampli-fied by self designed primer. The amplified gene wascloned in pET32(a) and expressed in BL21(DE3)PLysS(Novagen). The clones maintained as glycerated stockwere revived by sub-culturing in Luria–Bertani (LB) brothsupplemented with ampicillin (100 �g/ml) and chloram-phenicol (34 �g/ml). For mass scale production, freshly

grown overnight cultures were inoculated in LB medium(1000 ml) and incubated at 37 ◦C with shaking. The cellswere induced with 1 mM IPTG and incubated at 37 ◦C withshaking.

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.3.2. Purification and quantification of recombinantroteins

To purify the expressed proteins, the cell pellets werendividually resuspended in lysis buffer [8 M urea, 0.1 Ma2HPO4, 0.01 M Tris–Cl (pH 8.0) containing with 5–10 mM

midazole] at 4 ml per gram of wet weight of cells andixed by vortexing. The suspensions of cells were stirred

or 2 h at 22 ◦C in the shaking incubator at 220 rpm tonsure complete lysis. The cell lysates were centrifuged at0,000 rpm for 20 min at 4 ◦C to pellet the cellular debris.he supernatants containing the solubilized proteins wereubjected to purification by Ni-NTA agarose resin (Qia-en, Germany). Three different columns containing one mlach of the 50% Ni-NTA slurry was added to 4 ml of lysatend mixed gently by shaking for 30 min at room temper-ture. The mixtures were loaded into respective columnsnd the flow rate was adjusted to 0.5 ml/min. The columnsere washed with wash buffer (8 M urea, 0.1 M NaH2PO4,

.01 M Tris–Cl, pH 6.3, containing 5 mM imidazole). Boundon specific bacterial proteins were eluted using elutionuffer I, pH 5.9 (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris–Cl).he expressed proteins were eluted by buffer II, pH 4.58 M urea, 0.1 M NaH2PO4, 0.01 M Tris–Cl), fractions wereooled and dialyzed using gradient concentration of ureaor 72 h and finally the proteins were equilibrated withBS pH 7.4. The proteins were resolved in SDS-PAGE alongith bovine serum albumin (BSA) in the concentrations

f 1–10 �g per 20 �l of buffer. The band thickness of pro-ein sample matching with a particular concentration ofSA was used to calculate the concentration of the sample.he protein samples were labeled, mixed with cocktails ofrotease inhibitors (Amresco, USA) and stored at −20 ◦C.he expression of targeted proteins viz., rSPAG1, rTaSPnd rHaa86 was confirmed by western blotting using rab-it hyper immune serum raised against sporozoites of T.nnulata, T. annulata infected calf serum and anti-H. a. ana-olicum larval antibodies, respectively.

.4. Adjuvant

Montanide 888 in mineral oil (10% suspension) was useds an adjuvant for immunization of calves. Each dose of thentigens was diluted with 1 ml of PBS and mixed with equaluantity of adjuvant until a stable emulsion was obtained.

.5. Immunization of animals

The immunization experiment was performed on fiveroups of calves and details of schedule, dose and deliveryystem are given in Table 1.

.6. Challenge study

Fourteen days after the last immunization, each calf ofroups 1, 2 and 3 was challenged with eight T. annulatanfected adults of H. a. anatolicum. Each calf of groups 1,, 4 and 5 were challenged with fifty uninfected adults of

oth sexes (male and females in 1:1 ratio). The ticks wereut in cloth bags and tied to the ear pinna for feeding. Thengorged female ticks dropped in the bag were collectednd entomological parameters were recorded and analyzed

ology 188 (2012) 231– 238 233

as per the formula given by Fragoso et al. (1998) with minormodifications.

DT% = 100[

1 −(

NTVNTC

)]

where DT (%) is the percent reduction of challenged adults;NTV, mean number of adults dropped from the immunizedgroup of animals; NTC, mean number of adults droppedfrom the control group of animals.

DR(%) = 100[

1 −(

PMTVPMTC

)]

where DR (%) is percentage reduction of mean weight ofadult females; PMTV, mean weight of adult females fed onimmunized group of animals; PMTC, mean weight of adultfemales fed on control group of animals.

DO(%) = 100[

1 −(

PATVPATC

)]

where DO (%) is the percentage reduction of mean eggmasses; PATV, mean egg masses laid by the ticks fed onthe animals of immunized groups; PATC, mean egg masseslaid by the ticks fed on the animals of control group.

E(%) = 100[1 − (CRT × CRO)]

where E (%) is the efficacy of immunogens; CRT, the reduc-tion in the number of challenged adult females (NTV/NTC);CRO, the reduction in egg laying capacity of ticks fed onimmunized and control animals (PATV/PATC).

The efficacy percentage of rHaa86 was determined asfollows:

1) Comparing the group 1 with the adjuvant control group3.

2) Comparing the group 4 with PBS control group 5.

2.7. Clinical and serological monitoring of animals

Following challenge, the calves of groups 1, 2 and 3were monitored daily for rectal temperature. Examina-tion of submandibular lymph nodes for assessment ofenlargement and examination of stained biopsy smear oflymph node was performed from day 4 post-challenge (PC).Stained blood smears were examined regularly to estimatepiroplasm parasitaemia. The estimation of clinical sever-ity of theileriosis was based on the presence of sustainedfever (≥39.5 ◦C), macroschizont index (percentage of lym-phocytes infected with macroschizonts) in lymph glandbiopsy, percentage of erythrocytic parasitaemia and mor-tality. Post mortem lesions of animals which died due tolethal T. annulata challenge were recorded.

Blood samples were collected aseptically from all thecalves during pre and post tick challenge period at regu-lar intervals. Sera were separated and stored at −20 ◦C tillfurther use. A separate heparinized blood samples fromcalves of group 1, 2 and 3 were also collected twice aweek to record the percentage of packed cell volume (PCV)

and total leukocyte count (TLC) after immunization andchallenge. The antibody responses to rHaa86, rSPAG1 andrTaSP antigens were monitored by indirect ELISA. Initiallycheckerboard titration was used to optimize the reagents.

234 L. Jeyabal et al. / Veterinary Parasitology 188 (2012) 231– 238

Table 1Immunization schedule and doses of recombinant proteins in experimental calves.

Groupa Primaryb (0 day) 1st boosterb (30th days) 2nd boosterb (60th days)

1 400 �g rHaa86 400 �g rHaa86 100 �g rHaa862 400 �g rSPAG1 + 400 �g rTaSP 400 �g rSPAG1 + 400 �g rTaSP 100 �g rSPAG1 + 100 �g rTaSP3 Adjuvant Adjuvant Adjuvant4 400 �g rHaa86 400 �g rHaa86 100 �g rHaa865 PBS PBS PBS

a Cattle were randomly assigned to experimental groups (n = 6), groups 1, 2 and 3 were experimentally challenged with T. annulata infected ticks. Animals of H. a. alenge.

of all groups (except group 2) were challenged with T. annulata free adultsduring pre- and post immunization and for entomological data after chal

b Deep intra muscular injection in glutial muscle.

After optimization, 100 �l of each of the antigens in coatingbuffer was applied to the microtitre plates at a concen-trations of 6 �g/ml rHaa86, 1 �g/ml rSPAG1 and 4 �g/mlrTaSP The collected sera (primary antibody) were diluted1:100 with PBS and were used in triplicate wells for eachantigen. Anti-bovine peroxidase conjugate (Sigma Chem-ical Company, USA) was used at a dilution of 1:10,000 assecondary antibody and o-phenylenediamine dihydrochlo-ride (OPD) in phosphate citrate buffer was used for colourdevelopment. The reaction was stopped with 50 �l 3 N HClper well, and absorbance was recorded in an ELISA reader(Tecan-Sunrise, Austria) at 492 nm.

2.8. Statistical analysis

The analysis of variance was used for comparing theclinical data amongst the 1st, 2nd and 3rd groups ofthe experimental calves and humoral antibody responsesamong the different groups of the experimental calvesand between different days within the same group ofcalves. The entomological data after challenge infestationwere analyzed by similar method. Significance at 5% level(p < 0.05) was used to define differences in different param-eters.

3. Results

3.1. Characterization of recombinant proteins

The expression of the antigen was confirmed by SDS-PAGE in which the rHaa86, rSPAG1 and rTaSP weremigrated as 97, 34 and 40 kDa, respectively (Fig. 1A). In thewestern blot format, using primary antibodies, strong sig-nals were detected against rHaa86, rSPAG1 and rTaSP whenprobed with anti-H. a. anatolicum larval antibodies, anti-T. annulata sporozoites antibodies and T. annulata infectedcalf serum, respectively (Fig. 1B).

3.2. Effect of immunization

3.2.1. Pathogenicity of T. annulata in calvesAll the eighteen calves in groups 1, 2 and 3 developed

classical symptoms of theileriosis (Table 2) after challengewith infected ticks. However, the macroschizont index and

percentage of erythrocytic parasitaemia was less in rHaa86immunized groups of animals compared to groups 2 and 3.The mean time to detect fever in the calves and the timeto detect the intralymphocytic macroschizonts in lymph

natolicum. Animals were monitored for clinical and serological responses

nodes were significantly less (p < 0.01) in the calves of adju-vant control group (group 3). All the calves of groups 2 and 3died due to theileriosis on day 11 to day 23 post-challenge.The death of all calves of control group indicated thatthe sporozoite challenge was lethal. Postmortem exami-nation of the calves revealed enlargement of spleen andlymph glands and stained impression smears of lymphglands showed presence of numerous intralymphocyticmacroschizonts. Prominent abomasal ulcers were recordedin the calves of immunized groups which succumbed dueto theileriosis. In contrast, three calves of group 1 recoveredafter showing remission of fever on third week follow-ing challenge indicating protection conferred by rHaa86.The mean maximum macroschizont infection in the lymphgland (7.8% in group 1; 10.3% in group 2; 10.6% in group3) and erythrocytic parasitaemia (22.8% in group 1; 25.0%in group 2; 28.8% in group 3) were concordant with theprotective trend but the values were not statistically sig-nificant. The mean minimum PCV of calves of groups 2 and3 was significantly higher in comparison to calves of group1.

3.3. Feeding and reproductive performances ofchallenged ticks

In all the group of calves the adult ticks started feed-ing within 48 h of release. The mean difference in numberof ticks dropped, engorgement weight and the mean eggmasses (mg) laid by the ticks dropped from calves of dif-ferent groups were compared separately and presented inTable 3. There is a significant difference (p < 0.05) in themean number of engorged ticks dropped, mean engorge-ment weight, and mean egg masses laid by the ticksdropped from immunized groups (1 and 4) of animals incomparison to animals of groups 3 and 5, respectively. Thedirect effect of immunization of animals with rHaa86 onthe challenged ticks was 64.7% and 58.4%, respectively, fedon groups 1 and 4. The other entomological parameters viz.,DO%, DR% and E% were 43.6, 30.6 and 75.5 for the ticks fedon group 1 animals while the same data were 38.1, 31.2and 71.4 for the ticks fed on group 4 animals.

3.4. Immunological responses

The antibody responses against recombinant proteinswere monitored and presented in Figs. 2 and 3. Following2nd booster a significantly (p < 0.05) high mean antibodyresponse (OD = 8.5 times to pre immunization) against

L. Jeyabal et al. / Veterinary Parasitology 188 (2012) 231– 238 235

Fig. 1. The SDS-PAGE and western blot analysis of recombinant proteins. (A) Coomassie Brilliant Blue stained SDS-PAGE profile of expressed rHaa86(97 kDa), rSPAG1 (34 kDa) and rTaSP (40 kDa) under reducing conditions, collected at Ni-NTA purification process. M, protein molecular weight marker. (B)Western blot analysis of rHaa86, rSPAG1 and rTaSP protein probed with anti-Hyalomma larval antibodies, rabbit hyperimmune serum raised against GUTSand T. annulata infected calf serum, respectively.

Table 2Clinical data (mean ± SE) of immunized calves infected with T. annulata.

Groups Pyrexiaa MSb Piroplasmc MS%d Piroplasm (%)e WBCf (103 ml−1) PCV (%)g SRA

1 7.3 ± 0.35 8.1 ± 0.43 10.8 ± 0.23 7.8 ± 1.8 22.8 ± 4.0 3.4 ± 0.4 20.7 ± 1.3 3/62 6.8 ± 0.9 6.2 ± 0.43 10.1 ± 0.43 10.3 ± 1.9 25.0 ± 2.1 3.8 ± 0.9 24.9* ± 1.3 0/63 6.0* ± 0.33 6.1* ± 0.3 10.0 ± 0.5 10.6 ± 1.2 28.8 ± 9.7 4.03 ± 0.7 26.2* ± 2.7 0/6

MS, macroschizont; SRA, survival rate of animals.a Days post challenge (PC) when pyrexia was detected.b Days PC when intralymphocytic macroschizont (lymph node) was detected.c Days PC when intraerythrocytic piroplasm was detected.d Maximum percentage of intralymphocytic macroschizont infection in lymph node.e Maximum percentage of intraerythocytic piroplasm parasitaemia.f Minimum white blood cell (WBC) count.g Minimum percentage of packed cell volume (PCV).* (p < 0.05) in comparison to group 1 calves.

0

0.2

0.4

0.6

0.8

1

0 21 45 73 80 87 94 120115105

Days of blood collections

OD

at

49

2 n

m

Group 1-rHaa86 Group2 rTaSP

Group 2-rSPAG1 Group 3-Adjuvant

1st dose1st

booster

2nd

booster

Tick

infestations

Fig. 2. Pooled IgG antibody response of groups of experimental calves immunized with recombinant antigens viz., rHaa86, rTaSP, rSPAG1 and adjuvantcontrol. All the animals were challenged with eight T. annulata infected adults and animals of groups 1 and 3 were also challenged with uninfected adults ofH. a. anatolicum. The ELISA value for groups 2 and 3 was not presented beyond days 94, as animals died on day 85–97 post primary immunization. The 50%animals of group 1 were died on day 85–97 post primary immunization and mean antibody response was calculated accordingly. The time of vaccinationshots and tick infestations are indicated (arrow).

236 L. Jeyabal et al. / Veterinary Parasitology 188 (2012) 231– 238

0

0.2

0.4

0.6

0.8

1

0 21 45 73 80 87 94 120115105

Days of blood collections

OD

at

492 n

m

Group 4-rHaa86 Group 5-PBS

1st dose1st

booster

2nd

booster

Tick

infestations

animal

Fig. 3. Pooled IgG antibody response of animals of groups 4 and 5. All thevaccination shots and tick infestations are indicated (arrow).

rHaa86 was detected in all the animals of group 1 (n = 6)and the response persisted for 94 days of primary immu-nization but there is a fall in mean antibody response after94 days to 120 days (OD = 8.0 times to pre immuniza-tion, n = 3) post primary immunization. Antibody responsesto the T. annulata proteins (rTaSP and rSPAG1) were alsoincreased (OD = 5.5 times to preimmunization) in the calvesof group 2 before challenge and the responses were slightlyincreased after challenge infestations. It is to be noted thatalthough anti-rTaSP and anti-rSPAG1 value has increasedsignificantly to the pre-immunization value, all animalsdied on or before 97 days following lethal challenge withT. annulata. In comparison to group 1, the overall trend inmean antibody response to rHaa86 was higher in group 4animals than animals of group 1 which might be due to theanimals are free from T. annulata infection. There was noantibody response (OD492 = 0.065 ± 0.002–0.072 ± 0.004)against rHaa86 in control calves throughout the experi-mental period.

4. Discussion

An important impact of controlling tick infestations isthe reduction of transmission of economically importanttick-borne pathogens. The development of tick vaccineswith the dual effect of reduction of tick infestations and

Table 3Reduction in feeding and reproductive performances of adults of H. a. anatolicum

Experimental groupa Percent reduction (vaccinated/co

DT DO

Animals challenged with T. annulata infected and non-infected ticksrHaa86 (Group 1) 64.7% (6.0 ± 1.0)* 43.Adjuvant control (Group 3) (17.0 ± 5.0) (21

Animals challenged with non-infected ticksrHaa86 (Group 4) 58.4% (7.4 ± 1.9)* 38.PBS negative control (Group 5) (17.8 ± 4.0) (20

a Cattle were randomly assigned to experimental groups (n = 6), vaccinated andb The percent reduction was calculated with respect to the control group: DT

reduction in egg masses. In parenthesis are shown the average ± S.E. for adult femcompared by ANOVA with unequal variance between vaccinated and control gro

c Vaccine efficacy (E) was calculated as 100[1 − (CRT × CRO)], where CRT and Cas compared to the control group, respectively.

* p < 0.05.

s were challenged with uninfected adults of H. a. anatolicum. The time of

the incidence of tick-borne diseases while minimizing aca-ricide applications is essential towards improvement ofcattle health and production in tropical and subtropicalregions of the world. Although the efforts to develop vac-cines against tick-borne pathogens constitute a separateresearch focus, targeting both tick vector and pathogenwill probably be a feasible and productive strategy in theintegrated control programme. For example, the efficacy ofBm86 based vaccines in reducing clinical cases of babesio-sis, as well as tick infestations in vaccinated herds has beenestablished in extensive field trials (de la Fuente et al., 1998;Rodríguez Valle et al., 2004). In another experiment, Labudaet al. (2003) reported that vaccination of mice with theputative tick cement protein 64P prevented transmissionof TBE virus. Mice immunized with the recombinant 64Pantigen and challenge-exposed to infected Ixodes ricinushad reduced tick infestations and a higher rate of survival.This effect may be caused by a local inflammatory immuneresponse stimulated by tick feeding on 64P vaccinatedanimals that may partially abrogate modulation of host’simmune response by tick-secreted factors. While workingon subolesin, de la Fuente et al. (2005) have shown that

vaccination with recombinant subolesin affect the trans-mission of tick-borne pathogens by decreasing the vectorcapacity of ticks. In the present experiment, the antigenswere targeted on the basis of its established function to

on challenge infestations.

ntrol)b

DR Ec

6% (122.7 ± 12.7)* 30.6% (217.2 ± 9.4)* 75.5%7.4 ± 19.5) (312.8 ± 9.3)

1% (129.3 ± 13.0)* 31.2% (213.2 ± 10.9)* 71.4%9.0 ± 11.8) (309.7 ± 8.4)

challenged with H. a. anatolicum unfed adults., % reduction in tick infestation; DR, % reduction in tick weight; DO, %ale tick number, engorged tick weight (mg), egg masses (mg) and were

ups.RO are the reduction in the number of adult female ticks and egg masses

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L. Jeyabal et al. / Veterinary

ork against tick feeding and reproductive performancesr limiting the development of parasitic stages in the hostystem. For example, the efficacy of rHaa86 expressed inichia pastoris (Azhahianambi et al., 2009a) and in E. coliystem (Azhahianambi et al., 2009b) has been evaluatedgainst experimental homologus challenge infestationsnd 61.6–82.3% efficacy against vector ticks was obtained.imilarly, immunization of calves with SPAG-1 expresseds a fusion protein with a hepatitis virus B core antigenlicited partial protection in immunized calves (Boultert al., 1995). Immunization with different constructs ofPAG did not prevent onset of clinical theileriosis in calvespon challenge with virulent sporozoites. Further, there isn inverse relationship between survival rate in the immu-ized calves and the quantum of sporozoites in challenge

noculum (Boulter et al., 1995; Hall et al., 2000; Darghoutht al., 2006). The inclusion of antigens from schizont stagesas been emphasized to improve the level of protectionbtained by SPAG 1 (Boulter and Hall, 2000). The TaSPene encoded for macroschizont protein of T. annulata hav-ng high identity with N-and C-terminal regions of theolymorphic immunodominant antigen (PIM) and the exis-ence of a central polymorphic region suggested TaSP ashe T. annulata homologue of PIM (Schnittger et al., 2002).lthough, TaSP protein has been extensively tested for itsiagnostic potentiality (Salih et al., 2005; Seitzer et al.,007), the protein has not been tested for its protectivefficacy.

The death of all calves of control group due to theilerio-is confirmed the lethality of the dose used for challenge.he uniformity in lethal challenge was assured by chal-enging the animals with T. annulata infected unfed adults

hich were fed at nymphal stage on animals infected withiroplasms of T. annulata. The clinical picture of T. annulatahallenged calves was presented by pyrexia, intralym-hocytic macroschizont, intraerythrocytic piroplasm, WBCount and PCV. The early symptoms of pyrexia and detec-ion of intralymphocytic macroschizont in groups 2 and 3nimals in comparison to rHaa86 immunized calves (Group) indicated early parasitaemia development because of

ow rejection of T. annulata infected ticks. The mean min-mum PCV value of rHaa86 immunized (group 1) calvess lower because these animals survived longer than thenimals of groups 2 and 3, thereby there was more timeor the PCV value to decrease. Immunization with cock-ails of rSPAG1 and rTaSP did not protect the calves againstethal challenge of theileriosis. The eventual protection of0% immunized calves of group 1 against theileriosis coulde due to the effect of rHaa86 directly on reduction inumbers of ticks successfully fed on animals of immunizedroups. Successful transmission of Theileria depends on theuantum of doses of sporozoites in the saliva of the feed-

ng ticks. The sporogony in the unfed adult ticks stops at certain stage and resumes at a rapid rate when the ticktarts to feed (Young et al., 1979). In the present study, theartial disruption in the feeding of the ticks due to host’s

mmunity was reflected by the reductions in the dropping

ate and engorgement weight of the ticks fed on calves ofroup 1 (Table 3). The reduction in the egg masses coulde due to the malnutrition caused by improper feedingf the ticks fed on immunized calves. These events were

ology 188 (2012) 231– 238 237

expected to inhibit maturation of sporozoites and subse-quent introduction of low quantum of Theileria infectionby the ticks to immunized calves. The observations were inagreement with the previous report of Fivaz et al. (1989)who reported survivability of animals following immuniza-tion against Rhipicephalus appendiculatus and challengedby tick induced T. parva infection.

Following immunization of animals with rHaa86, anincrease in rejection percentage and decrease in repro-ductive index of challenged ticks fed on the immunizedanimals of groups 1 and 4 were recorded and the datawere comparable with Bm86 (GavacTM) vaccine. The E%of the Gavac vaccine against larvae of different strains ofR. (B.) microplus varied from 51 to 91%. In the presentstudy, 71.4–75.5% efficacy was obtained by challenging theimmunized calves with adults of H. a. anatolicum and theefficacy percentage is falling within the range of the workreported by Canales et al. (1997). The DT% of the GavacTM

vaccine against different strains of R. (B.) microplus was9–74%. Amongst the ten strains, DT% of above 50% wasrecorded against two strains. The average DT% obtained inthe present study was comparable with the DT% obtainedusing GavacTM vaccine (Canales et al., 1997). In the presentexperiment, no improvement in survival rate after a lethalchallenge with sporozoites was observed when rTaSP wereincorporated with rSPAG1 for immunization. Moreover,enhanced protection against theileriosis could be expectedby rHaa86 antigen in actual field conditions prevailing inIndia where high quantum of natural challenge with T.annulata is rare because of enzootic stability (Das and Ray,2003). The experiment has given a significant clue towardsthe development of rHaa86 based vaccine against both H.a. anatolicum and T. annulata.

Acknowledgements

Sincere thanks are due to Department of Biotechnol-ogy Government of India for funding the project (No.BT/PR6177/AAQ/01/232/2005). This work has been facil-itated through the Integrated Consortium on Ticks andTick-borne Diseases (ICTTD-3), financed by the Interna-tional Cooperation Programme of the European Unionthrough Coordination Action Project no. 510561. The con-tribution made by the laboratory staff (Mr. Laxmi Lal,Naresh Kumar and Mohan Lal) is highly acknowledged.

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