Vaccine potential of recombinant antigens of Theileria annulata and Hyalomma anatolicum anatolicum against vector and parasite

Veterinary Parasitology 188 (2012) 231–238

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Vaccine potential of recombinant antigens of Theileria annulata and Hyalomma anatolicum anatolicum against vector and parasite L. Jeyabal a , Binod Kumar a , Debdatta Ray a , Palavesam Azahahianambi b , Srikanta Ghosh a,∗ a b

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

a r t i c l e

i n f o

Article history: Received 15 October 2011 Received in revised form 26 March 2012 Accepted 28 March 2012 Keywords: rHaa86 rSPAG1 rTaSP Hyalomma anatolicum anatolicum Theileria annulata

a b s t r a c t In an attempt to develop vaccine against Hyalomma anatolicum anatolicum and Theileria annulata, three antigens were expressed in prokaryotic expression system and protective potentiality 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 Bm86 ortholog of H. a. anatolicum, while one group of calves (gr. 2) were immunized with cocktails of two antigens viz., surface antigens of T. annulata (rSPAG1, rTaSP). One group each was kept as negative controls (grs. 3 and 5). The animals of groups 1, 2 and 3 were challenged with T. annulata infected H. a. anatolicum adults while the animals of groups 1, 3, 4 and 5 were challenged with uninfected adult ticks. A significantly high (p < 0.05) antibody responses to all the three antigens were detected in immunized calves, but the immune response was comparatively 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 to protect calves against more than 71.4–75.5% of the challenge infestation. The experiment has given a significant clue towards the development of rHaa86 based vaccine against both H. a. anatolicum and T. annulata. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Tick infestations significantly impact cattle production by reducing weight gain, milk production and also by transmitting pathogens (Peter et al., 2004). In India, out of 106 reported tick species infesting animals, Hyalomma anatolicum anatolicum, the vector of the apicomplexan parasite, Theileria annulata, is distributed widely (Ghosh et al., 2007). Tropical theileriosis caused by T. annulata affect cattle in a large geographical areas of north Africa, southern Europe and most of the parts of Asia. In India, this vector–pathogen combinations is a significant contributor

∗ Corresponding author. Tel.: +91 581 2302368; fax: +91 941 0261029. E-mail address: [email protected] (S. Ghosh). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.03.051

to control cost incurred in the tune of 498.7 million US$ per annum (Minjauw and McLeod, 2003). Current control of H. a. anatolicum and T. annulata largely focused on repeated use of acaricides which has limited efficacy and is often accompanied by serious drawbacks viz., selection of acaricides-resistant ticks, environmental pollution and contamination of livestock products with acaricide residues (Graf et al., 2004). Other methods of tick control include the use of hosts with natural 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) microplus using selected antigens (Bm86) was demonstrated and vaccines have been developed for successful control of the tick species (de la Fuente and Kocan, 2006; Willadsen,

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2006; de la Fuente et al., 2007). The ortholog gene of Bm86 was identified in other tick species and possibilities of development of recombinant vaccines against these tick species have been explored (de la Fuente and Kocan, 2003; Azhahianambi et al., 2009a; Nijhof et al., 2010). Immunological control of bovine tropical theileriosis is achieved by the use of attenuated schizont vaccine in Israel (Pipano and Tsur, 1966), Iran (Hashemi-Fesharky, 1988), Turkey (Sayin et al., 1997), Tunisia (Darghouth, 2008), China (Zhang, 1997) and in India (Subramanian et al., 1988). However, the vaccine has limitations like the requirement of cold chain for preservation and the possibility of reversion of the attenuated strain to virulent strain. To overcome these problems, research for development of subunit vaccine against T. annulata infection had been focused on surface antigens present on sporozoites and merozoites (Boulter and Hall, 2000). Immunization of animals with different constructs of sporozoite surface protein (SPAG1) provided partial protection in calves probably through reduction in the number of sporozoites in challenge infection and their reduced ability to invade target host cells (Boulter et al., 1998). Inclusion of more antigens 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 vector ticks was shown to be effective in reducing the ability of ticks to transmit Theileria parva (Fivaz et al., 1989), T. annulata (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 identified in H. a. anatolicum having seven complete EGF-like domain and the antigen was found protective in conferring protection against experimental challenge infestations (Azhahianambi et al., 2009a). In the present study, attempt was made to evaluate the protective efficacy of rHaa86 against the homologus challenge infestations and against lethal dose of T. annulata. The result was compared with the efficacy of a cocktail of recombinant proteins of T. annulata (SPAG1 and TaSP) in conferring protection against theileriosis 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 institute dairy farm. The calves were housed individually in tick proof pens and maintained on milk, grain concentrates and wheat bhusa. The blood smears of the calves were examined for the presence of any hemoparasites and their serum samples were examined for antibodies against soluble piroplasm antigen of T. annulata or larval antigen of H. a. anatolicum to ensure freedom from any previous exposures to vector and parasites. The experimental animals were maintained as per the approved guidelines laid down by the committee for the purpose of control and supervision of experimental animals (CPCSEA), a statutory Indian body.

2.2. Ticks 2.2.1. T. annulata free adult H. a. anatolicum ticks The homogenous T. annulata free H. a. anatolicum, IVRI line II was maintained in the Entomology laboratory of the Division of Parasitology as per standardized protocol (Ghosh and Azhahianambi, 2007). Healthy New Zealand white rabbits weighing 1.5–2 kg were used for feeding of larvae of tick species. The adults were fed on disease free male cross bred calves. The engorged adults were kept singly in tick rearing glass vials maintained at 28 ◦ C and 85% RH in BOD incubator for egg laying.

2.2.2. T. annulata infected adults of H. a. anatolicum Two four months old cross bred male calves were inoculated subcutaneously with a cryopreserved stabilate of T. annulata (Parbhani isolate) equivalent to 2 infected ticks. Uninfected nymphs were allowed to feed on the infected calves when intra-erythrocytic piroplasms were detected in the blood smears (showing 30% of RBCs infected with piroplasms of T. annulata). The fully engorged nymphs were collected, reared in the laboratory. The molted adults were reared for seven to ten days before used for challenging immunized and control calves.

2.3. Recombinant antigens 2.3.1. Mass culture of recombinant bacterial clones rSPAG1: The details of expression of SPAG1 gene are elaborated in Vanlalmuaka et al. (2010). The gene was cloned in expression vector pET32c(+) and transformed into a BL21(DE3) pLysS strain of Escherichia coli. Expression was induced with 1 mM IPTG and incubated at 37 ◦ C in shaking incubator. The expression was confirmed by SDSPAGE. rTaSP: The details of expression of the gene are elaborated in Vanlalmuaka et al. (2010). In brief, the gene coding for 26–156 amino acids of the TaSP gene was PCR amplified using custom synthesized oligonucleotide primers: 5 F GTC GAC CAT GGA TCG ACA ACT TAA TCC 3 and 5 R ATC TGC AGT ACC CGT CAG ACT CAT CAT C 3 . The amplified gene was cloned into pET32c(+) (Novagen, Merck Biosciences, Germany) for expression in BL21(DE3)pLysS E. coli. Expression was induced with IPTG and expression profile was analyzed under denaturing condition. rHaa86: The details of expression of the gene are elaborated in Azhahianambi et al. (2009a,b). On the basis of published sequence information (AF347079), a 1.755 kb size Bm86 ortholog gene (Haa86) was amplified by self designed primer. The amplified gene was cloned in pET32(a) and expressed in BL21(DE3)PLysS (Novagen). The clones maintained as glycerated stock were revived by sub-culturing in Luria–Bertani (LB) broth supplemented with ampicillin (100 ␮g/ml) and chloramphenicol (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 cells were induced with 1 mM IPTG and incubated at 37 ◦ C with shaking.

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2.3.2. Purification and quantification of recombinant proteins To purify the expressed proteins, the cell pellets were individually resuspended in lysis buffer [8 M urea, 0.1 M Na2 HPO4 , 0.01 M Tris–Cl (pH 8.0) containing with 5–10 mM imidazole] at 4 ml per gram of wet weight of cells and mixed by vortexing. The suspensions of cells were stirred for 2 h at 22 ◦ C in the shaking incubator at 220 rpm to ensure complete lysis. The cell lysates were centrifuged at 10,000 rpm for 20 min at 4 ◦ C to pellet the cellular debris. The supernatants containing the solubilized proteins were subjected to purification by Ni-NTA agarose resin (Qiagen, Germany). Three different columns containing one ml each of the 50% Ni-NTA slurry was added to 4 ml of lysate and mixed gently by shaking for 30 min at room temperature. The mixtures were loaded into respective columns and the flow rate was adjusted to 0.5 ml/min. The columns were washed with wash buffer (8 M urea, 0.1 M NaH2 PO4 , 0.01 M Tris–Cl, pH 6.3, containing 5 mM imidazole). Bound non specific bacterial proteins were eluted using elution buffer I, pH 5.9 (8 M urea, 0.1 M NaH2 PO4 , 0.01 M Tris–Cl). The expressed proteins were eluted by buffer II, pH 4.5 (8 M urea, 0.1 M NaH2 PO4 , 0.01 M Tris–Cl), fractions were pooled and dialyzed using gradient concentration of urea for 72 h and finally the proteins were equilibrated with PBS pH 7.4. The proteins were resolved in SDS-PAGE along with bovine serum albumin (BSA) in the concentrations of 1–10 ␮g per 20 ␮l of buffer. The band thickness of protein sample matching with a particular concentration of BSA was used to calculate the concentration of the sample. The protein samples were labeled, mixed with cocktails of protease inhibitors (Amresco, USA) and stored at −20 ◦ C. The expression of targeted proteins viz., rSPAG1, rTaSP and rHaa86 was confirmed by western blotting using rabbit hyper immune serum raised against sporozoites of T. annulata, T. annulata infected calf serum and anti-H. a. anatolicum larval antibodies, respectively.

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

2.4. Adjuvant

Following challenge, the calves of groups 1, 2 and 3 were monitored daily for rectal temperature. Examination of submandibular lymph nodes for assessment of enlargement and examination of stained biopsy smear of lymph node was performed from day 4 post-challenge (PC). Stained blood smears were examined regularly to estimate piroplasm parasitaemia. The estimation of clinical severity of theileriosis was based on the presence of sustained fever (≥39.5 ◦ C), macroschizont index (percentage of lymphocytes infected with macroschizonts) in lymph gland biopsy, percentage of erythrocytic parasitaemia and mortality. Post mortem lesions of animals which died due to lethal T. annulata challenge were recorded. Blood samples were collected aseptically from all the calves during pre and post tick challenge period at regular intervals. Sera were separated and stored at −20 ◦ C till further use. A separate heparinized blood samples from calves of group 1, 2 and 3 were also collected twice a week to record the percentage of packed cell volume (PCV) and total leukocyte count (TLC) after immunization and challenge. The antibody responses to rHaa86, rSPAG1 and rTaSP antigens were monitored by indirect ELISA. Initially checkerboard titration was used to optimize the reagents.

Montanide 888 in mineral oil (10% suspension) was used as an adjuvant for immunization of calves. Each dose of the antigens was diluted with 1 ml of PBS and mixed with equal quantity of adjuvant until a stable emulsion was obtained. 2.5. Immunization of animals The immunization experiment was performed on five groups of calves and details of schedule, dose and delivery system are given in Table 1. 2.6. Challenge study Fourteen days after the last immunization, each calf of groups 1, 2 and 3 was challenged with eight T. annulata infected adults of H. a. anatolicum. Each calf of groups 1, 3, 4 and 5 were challenged with fifty uninfected adults of both sexes (male and females in 1:1 ratio). The ticks were put in cloth bags and tied to the ear pinna for feeding. The engorged female ticks dropped in the bag were collected and entomological parameters were recorded and analyzed



DT% = 100 1 −

 NTV  NTC

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



DR(%) = 100 1 −

 PMTV  PMTC

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



DO(%) = 100 1 −

 PATV  PATC

where DO (%) is the percentage reduction of mean egg masses; PATV, mean egg masses laid by the ticks fed on the animals of immunized groups; PATC, mean egg masses laid by the ticks fed on the animals of control group. E(%) = 100[1 − (CRT × CRO)] where E (%) is the efficacy of immunogens; CRT, the reduction in the number of challenged adult females (NTV/NTC); CRO, the reduction in egg laying capacity of ticks fed on immunized and control animals (PATV/PATC). The efficacy percentage of rHaa86 was determined as follows: 1) Comparing the group 1 with the adjuvant control group 3. 2) Comparing the group 4 with PBS control group 5. 2.7. Clinical and serological monitoring of animals

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

Primaryb (0 day)

1st boosterb (30th days)

2nd boosterb (60th days)

1 2 3 4 5

400 ␮g rHaa86 400 ␮g rSPAG1 + 400 ␮g rTaSP Adjuvant 400 ␮g rHaa86 PBS

400 ␮g rHaa86 400 ␮g rSPAG1 + 400 ␮g rTaSP Adjuvant 400 ␮g rHaa86 PBS

100 ␮g rHaa86 100 ␮g rSPAG1 + 100 ␮g rTaSP Adjuvant 100 ␮g rHaa86 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 all groups (except group 2) were challenged with T. annulata free adults of H. a. anatolicum. Animals were monitored for clinical and serological responses during pre- and post immunization and for entomological data after challenge. b Deep intra muscular injection in glutial muscle.

After optimization, 100 ␮l of each of the antigens in coating buffer was applied to the microtitre plates at a concentrations of 6 ␮g/ml rHaa86, 1 ␮g/ml rSPAG1 and 4 ␮g/ml rTaSP The collected sera (primary antibody) were diluted 1:100 with PBS and were used in triplicate wells for each antigen. Anti-bovine peroxidase conjugate (Sigma Chemical Company, USA) was used at a dilution of 1:10,000 as secondary antibody and o-phenylenediamine dihydrochloride (OPD) in phosphate citrate buffer was used for colour development. The reaction was stopped with 50 ␮l 3 N HCl per 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 the clinical data amongst the 1st, 2nd and 3rd groups of the experimental calves and humoral antibody responses among the different groups of the experimental calves and between different days within the same group of calves. The entomological data after challenge infestation were analyzed by similar method. Significance at 5% level (p < 0.05) was used to define differences in different parameters. 3. Results 3.1. Characterization of recombinant proteins The expression of the antigen was confirmed by SDSPAGE in which the rHaa86, rSPAG1 and rTaSP were migrated as 97, 34 and 40 kDa, respectively (Fig. 1A). In the western blot format, using primary antibodies, strong signals were detected against rHaa86, rSPAG1 and rTaSP when probed with anti-H. a. anatolicum larval antibodies, antiT. annulata sporozoites antibodies and T. annulata infected calf serum, respectively (Fig. 1B). 3.2. Effect of immunization 3.2.1. Pathogenicity of T. annulata in calves All the eighteen calves in groups 1, 2 and 3 developed classical symptoms of theileriosis (Table 2) after challenge with infected ticks. However, the macroschizont index and percentage of erythrocytic parasitaemia was less in rHaa86 immunized groups of animals compared to groups 2 and 3. The mean time to detect fever in the calves and the time to detect the intralymphocytic macroschizonts in lymph

nodes were significantly less (p < 0.01) in the calves of adjuvant control group (group 3). All the calves of groups 2 and 3 died due to theileriosis on day 11 to day 23 post-challenge. The death of all calves of control group indicated that the sporozoite challenge was lethal. Postmortem examination of the calves revealed enlargement of spleen and lymph glands and stained impression smears of lymph glands showed presence of numerous intralymphocytic macroschizonts. Prominent abomasal ulcers were recorded in the calves of immunized groups which succumbed due to theileriosis. In contrast, three calves of group 1 recovered after showing remission of fever on third week following challenge indicating protection conferred by rHaa86. The mean maximum macroschizont infection in the lymph gland (7.8% in group 1; 10.3% in group 2; 10.6% in group 3) and erythrocytic parasitaemia (22.8% in group 1; 25.0% in group 2; 28.8% in group 3) were concordant with the protective trend but the values were not statistically significant. The mean minimum PCV of calves of groups 2 and 3 was significantly higher in comparison to calves of group 1. 3.3. Feeding and reproductive performances of challenged ticks In all the group of calves the adult ticks started feeding within 48 h of release. The mean difference in number of ticks dropped, engorgement weight and the mean egg masses (mg) laid by the ticks dropped from calves of different groups were compared separately and presented in Table 3. There is a significant difference (p < 0.05) in the mean number of engorged ticks dropped, mean engorgement weight, and mean egg masses laid by the ticks dropped from immunized groups (1 and 4) of animals in comparison to animals of groups 3 and 5, respectively. The direct effect of immunization of animals with rHaa86 on the challenged ticks was 64.7% and 58.4%, respectively, fed on groups 1 and 4. The other entomological parameters viz., DO%, DR% and E% were 43.6, 30.6 and 75.5 for the ticks fed on group 1 animals while the same data were 38.1, 31.2 and 71.4 for the ticks fed on group 4 animals. 3.4. Immunological responses The antibody responses against recombinant proteins were monitored and presented in Figs. 2 and 3. Following 2nd booster a significantly (p < 0.05) high mean antibody response (OD = 8.5 times to pre immunization) against

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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 GUTS and T. annulata infected calf serum, respectively.

Table 2 Clinical 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 2 3

7.3 ± 0.35 6.8 ± 0.9 6.0* ± 0.33

8.1 ± 0.43 6.2 ± 0.43 6.1* ± 0.3

10.8 ± 0.23 10.1 ± 0.43 10.0 ± 0.5

7.8 ± 1.8 10.3 ± 1.9 10.6 ± 1.2

22.8 ± 4.0 25.0 ± 2.1 28.8 ± 9.7

3.4 ± 0.4 3.8 ± 0.9 4.03 ± 0.7

20.7 ± 1.3 24.9* ± 1.3 26.2* ± 2.7

3/6 0/6 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.

1st 1 dose booster st

2nd Tick booster infestations

OD at 492 nm

1 0.8 0.6 0.4 0.2 0 0

21

45

73

80

87

94

105

115

120

Days of blood collections Group 1-rHaa86 Group2 rTaSP Group 2-rSPAG1

Group 3-Adjuvant

Fig. 2. Pooled IgG antibody response of groups of experimental calves immunized with recombinant antigens viz., rHaa86, rTaSP, rSPAG1 and adjuvant control. All the animals were challenged with eight T. annulata infected adults and animals of groups 1 and 3 were also challenged with uninfected adults of H. 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 vaccination shots and tick infestations are indicated (arrow).

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1st 1 dose booster st

2nd Tick booster infestations

OD at 492 nm

1 0.8 0.6 0.4 0.2 0 0

21

45

73

80

87

94

105

115

120

Days of blood collections Group 4-rHaa86

Group 5-PBS

Fig. 3. Pooled IgG antibody response of animals of groups 4 and 5. All the animals were challenged with uninfected adults of H. a. anatolicum. The time of vaccination 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 immunization but there is a fall in mean antibody response after 94 days to 120 days (OD = 8.0 times to pre immunization, n = 3) post primary immunization. Antibody responses to the T. annulata proteins (rTaSP and rSPAG1) were also increased (OD = 5.5 times to preimmunization) in the calves of group 2 before challenge and the responses were slightly increased after challenge infestations. It is to be noted that although anti-rTaSP and anti-rSPAG1 value has increased significantly to the pre-immunization value, all animals died on or before 97 days following lethal challenge with T. annulata. In comparison to group 1, the overall trend in mean antibody response to rHaa86 was higher in group 4 animals than animals of group 1 which might be due to the animals are free from T. annulata infection. There was no antibody response (OD492 = 0.065 ± 0.002–0.072 ± 0.004) against rHaa86 in control calves throughout the experimental period. 4. Discussion An important impact of controlling tick infestations is the reduction of transmission of economically important tick-borne pathogens. The development of tick vaccines with the dual effect of reduction of tick infestations and

the incidence of tick-borne diseases while minimizing acaricide applications is essential towards improvement of cattle health and production in tropical and subtropical regions of the world. Although the efforts to develop vaccines against tick-borne pathogens constitute a separate research focus, targeting both tick vector and pathogen will probably be a feasible and productive strategy in the integrated control programme. For example, the efficacy of Bm86 based vaccines in reducing clinical cases of babesiosis, as well as tick infestations in vaccinated herds has been established in extensive field trials (de la Fuente et al., 1998; Rodríguez Valle et al., 2004). In another experiment, Labuda et al. (2003) reported that vaccination of mice with the putative tick cement protein 64P prevented transmission of TBE virus. Mice immunized with the recombinant 64P antigen and challenge-exposed to infected Ixodes ricinus had reduced tick infestations and a higher rate of survival. This effect may be caused by a local inflammatory immune response stimulated by tick feeding on 64P vaccinated animals that may partially abrogate modulation of host’s immune response by tick-secreted factors. While working on subolesin, de la Fuente et al. (2005) have shown that vaccination with recombinant subolesin affect the transmission of tick-borne pathogens by decreasing the vector capacity of ticks. In the present experiment, the antigens were targeted on the basis of its established function to

Table 3 Reduction in feeding and reproductive performances of adults of H. a. anatolicum on challenge infestations. Experimental groupa

Percent reduction (vaccinated/control)b DT

Animals challenged with T. annulata infected and non-infected ticks 64.7% (6.0 ± 1.0)* rHaa86 (Group 1) (17.0 ± 5.0) Adjuvant control (Group 3) Animals challenged with non-infected ticks 58.4% (7.4 ± 1.9)* rHaa86 (Group 4) (17.8 ± 4.0) PBS negative control (Group 5) a

DO

DR

Ec

43.6% (122.7 ± 12.7)* (217.4 ± 19.5)

30.6% (217.2 ± 9.4)* (312.8 ± 9.3)

75.5%

38.1% (129.3 ± 13.0)* (209.0 ± 11.8)

31.2% (213.2 ± 10.9)* (309.7 ± 8.4)

71.4%

Cattle were randomly assigned to experimental groups (n = 6), vaccinated and challenged with H. a. anatolicum unfed adults. The percent reduction was calculated with respect to the control group: DT, % reduction in tick infestation; DR, % reduction in tick weight; DO, % reduction in egg masses. In parenthesis are shown the average ± S.E. for adult female tick number, engorged tick weight (mg), egg masses (mg) and were compared by ANOVA with unequal variance between vaccinated and control groups. c Vaccine efficacy (E) was calculated as 100[1 − (CRT × CRO)], where CRT and CRO are the reduction in the number of adult female ticks and egg masses as compared to the control group, respectively. * p < 0.05. b

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work against tick feeding and reproductive performances or limiting the development of parasitic stages in the host system. For example, the efficacy of rHaa86 expressed in Pichia pastoris (Azhahianambi et al., 2009a) and in E. coli system (Azhahianambi et al., 2009b) has been evaluated against experimental homologus challenge infestations and 61.6–82.3% efficacy against vector ticks was obtained. Similarly, immunization of calves with SPAG-1 expressed as a fusion protein with a hepatitis virus B core antigen elicited partial protection in immunized calves (Boulter et al., 1995). Immunization with different constructs of SPAG did not prevent onset of clinical theileriosis in calves upon challenge with virulent sporozoites. Further, there is an inverse relationship between survival rate in the immunized calves and the quantum of sporozoites in challenge inoculum (Boulter et al., 1995; Hall et al., 2000; Darghouth et al., 2006). The inclusion of antigens from schizont stages has been emphasized to improve the level of protection obtained by SPAG 1 (Boulter and Hall, 2000). The TaSP gene encoded for macroschizont protein of T. annulata having high identity with N-and C-terminal regions of the polymorphic immunodominant antigen (PIM) and the existence of a central polymorphic region suggested TaSP as the T. annulata homologue of PIM (Schnittger et al., 2002). Although, TaSP protein has been extensively tested for its diagnostic potentiality (Salih et al., 2005; Seitzer et al., 2007), the protein has not been tested for its protective efficacy. The death of all calves of control group due to theileriosis confirmed the lethality of the dose used for challenge. The uniformity in lethal challenge was assured by challenging the animals with T. annulata infected unfed adults which were fed at nymphal stage on animals infected with piroplasms of T. annulata. The clinical picture of T. annulata challenged calves was presented by pyrexia, intralymphocytic macroschizont, intraerythrocytic piroplasm, WBC count and PCV. The early symptoms of pyrexia and detection of intralymphocytic macroschizont in groups 2 and 3 animals in comparison to rHaa86 immunized calves (Group 1) indicated early parasitaemia development because of low rejection of T. annulata infected ticks. The mean minimum PCV value of rHaa86 immunized (group 1) calves is lower because these animals survived longer than the animals of groups 2 and 3, thereby there was more time for the PCV value to decrease. Immunization with cocktails of rSPAG1 and rTaSP did not protect the calves against lethal challenge of theileriosis. The eventual protection of 50% immunized calves of group 1 against theileriosis could be due to the effect of rHaa86 directly on reduction in numbers of ticks successfully fed on animals of immunized groups. Successful transmission of Theileria depends on the quantum of doses of sporozoites in the saliva of the feeding ticks. The sporogony in the unfed adult ticks stops at a certain stage and resumes at a rapid rate when the tick starts to feed (Young et al., 1979). In the present study, the partial disruption in the feeding of the ticks due to host’s immunity was reflected by the reductions in the dropping rate and engorgement weight of the ticks fed on calves of group 1 (Table 3). The reduction in the egg masses could be due to the malnutrition caused by improper feeding of the ticks fed on immunized calves. These events were

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expected to inhibit maturation of sporozoites and subsequent introduction of low quantum of Theileria infection by the ticks to immunized calves. The observations were in agreement with the previous report of Fivaz et al. (1989) who reported survivability of animals following immunization against Rhipicephalus appendiculatus and challenged by tick induced T. parva infection. Following immunization of animals with rHaa86, an increase in rejection percentage and decrease in reproductive index of challenged ticks fed on the immunized animals of groups 1 and 4 were recorded and the data were comparable with Bm86 (GavacTM ) vaccine. The E% of the Gavac vaccine against larvae of different strains of R. (B.) microplus varied from 51 to 91%. In the present study, 71.4–75.5% efficacy was obtained by challenging the immunized calves with adults of H. a. anatolicum and the efficacy percentage is falling within the range of the work reported by Canales et al. (1997). The DT% of the GavacTM vaccine against different strains of R. (B.) microplus was 9–74%. Amongst the ten strains, DT% of above 50% was recorded against two strains. The average DT% obtained in the present study was comparable with the DT% obtained using GavacTM vaccine (Canales et al., 1997). In the present experiment, no improvement in survival rate after a lethal challenge with sporozoites was observed when rTaSP were incorporated with rSPAG1 for immunization. Moreover, enhanced protection against theileriosis could be expected by rHaa86 antigen in actual field conditions prevailing in India 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 towards the development of rHaa86 based vaccine against both H. a. anatolicum and T. annulata.

Acknowledgements Sincere thanks are due to Department of Biotechnology Government of India for funding the project (No. BT/PR6177/AAQ/01/232/2005). This work has been facilitated through the Integrated Consortium on Ticks and Tick-borne Diseases (ICTTD-3), financed by the International Cooperation Programme of the European Union through Coordination Action Project no. 510561. The contribution made by the laboratory staff (Mr. Laxmi Lal, Naresh Kumar and Mohan Lal) is highly acknowledged.

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