Efficacy of rHaa86, an Orthologue of Bm86, Against Challenge Infestations of Hyalomma anatolicum anatolicum

ORIGINAL ARTICLE

Efficacy of rHaa86, an Orthologue of Bm86, AgainstChallenge Infestations of Hyalomma anatolicumanatolicumL. Jeyabal, P. Azhahianambi, K. Susitha, D. D. Ray, P. Chaudhuri, Vanlahmuaka and S. Ghosh

Entomology Laboratory, Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India

Introduction

In India, almost all dairy and draft cattle are suffering from

tick infestations of different intensity (Ghosh et al., 2006)

and the impact of ticks and tick-borne diseases (TBD) on

livestock economy has recently been estimated in the tune

of 498.7 million US$/annum (Minjauw and McLeod,

2003). Of the 106 existing tick species reported from India,

Hyalomma anatolicum anatolicum is widely distributed

throughout India excepting few north eastern states

(Ghosh et al., 2007) and is targeted for control of bovine

tropical theileriosis. Apart from transmission of diseases,

the normal feeding activity of ticks on heavily infested

animals results in significant economic losses to animal

owners because of reduction in weight gain and milk pro-

duction and to leather sector which is suffering from huge

shortfall of good quality hides because of tick-bite marks

(Leatherware, 2002). The prevailing control method of

ticks is based on intensive use of acaricides is beset with

limitations like selection of chemical resistant ticks along

with contamination of environment and animal products.

To overcome the problems associated with the use of acari-

cides, immunization of the host was suggested as one of

the most cost-effective approach for the control of both

ticks and TBD (Willadsen, 2004; de la Fuente et al., 2007).

The protective ability of crude and partially purified

antigens of H. a. anatolicum was reported and the subject

has been recently reviewed (Ghosh et al., 2008). In the

Entomology Laboratory of Indian Veterinary Research

Institute Bm86 orthologue of H. a. anatolicum has recently

been cloned and expressed in Pichia pastoris and the

recombinant protein (rHa86) in combination with saponin

in mineral oil has been found protective against homolo-

gous challenge infestations (Azhahianambi et al., 2009).

The purification process of P. pastoris-expressed rHaa86

was very tedious and low expression of the protein has

Keywords:

efficacy; Hyalomma a. anatolicum;

immunization; rHaa86; Theileria annulata

Correspondence:

S. Ghosh, Entomology Laboratory, Division of

Parasitology, Indian Veterinary Research

Institute, Izatnagar, Bareilly, Uttar Pradesh,

India. Tel.: +91 581 230236; Fax: +91 581

230236; E-mail: [email protected]

Received for publication February 03, 2010

doi:10.1111/j.1865-1682.2010.01107.x

Summary

In an attempt to develop vaccine against Hyalomma anatolicum anatolicum, the

protective efficacy of rHaa86 was evaluated against experimental challenge

infestations of homologous tick species and lethal dose of Theileria annulata.

Following challenge, a significant difference of 20.9% (P < 0.01) in the drop-

ping per cent of ticks fed on immunized and control animals was recorded.

A statistically significant reduction of 49.6 mg (P < 0.01) in the weight of ticks

fed on immunized animals in comparison with control was noted. The ticks

dropped from immunized animals laid fewer eggs and a reduction of 68.1 mg

(P < 0.05) in comparison with the ticks fed on control animals was noted. The

DT%, DO%, DR% and E% were calculated as 73.8, 31.3, 15.8 and 82.3%

respectively. In all the calves fever (rectal temperature £39.5�C) was detected

after a mean period of 7.2 days in immunized calves and on 5.8 days in control

calves following lethal challenge with T. annulata. The mean Maximum Macro-

schizonts Index was 7.8% and 10.6% in the immunized and control calves

respectively. Two calves (identification no. 351 and 354) died in the immu-

nized group while all five calves died in the control group. The data demon-

strated that rHaa86 antigen-based vaccine could serve as one of the effective

components of the integrated control of H. a. anatolicum and T. annulata.

Transboundary and Emerging Diseases

96 ª 2010 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 57 (2010) 96–102

been reported. In continuation, attempts were made to

express rHaa86 in prokaryotic expression system with sig-

nificantly less steps in purification. The present experiment

was conducted to evaluate the efficacy of the prokaryotic

expressed rHaa86 against homologous challenge infesta-

tions and also against lethal dose of Theileria annulata.

Material and Methods

Experimental animals

Cross-bred calves

Cross-bred bovine calves (Bos tauras male · B. indicus

female) were used in the study. The animals were procured

from Livestock Production and Management Section,

Indian Veterinary Research Institute, Izatnagar just after

weaning and maintained in the tick proof shed of the

Division of Parasitology. All the animals were treated with

Albendazole (Albomol�, Albomar suspention; Glaxo Smith

Kline Pharmaceuticals Ltd., Brentford, Middlesex, UK)

15 mg/kg body weight orally. To maintain tick naı̈ve sta-

tus, all the animals were strategically treated with 0.05%

cypermethrin and the animal shed was treated with 0.5%

lindane. The application of all the insecticides was stopped

1 month before the start of the experiment. The animals

were fed on a daily ration of concentrate and wheat bran

with ad lib drinking water throughout the experimental

period. No green fodder was supplied to the animals to

avoid extraneous infestation of ticks. Each calf was injected

intramuscularly once with 600 000 IU of vitamin A. The

calves were approximately 7 months old when immunized.

Rabbits

New Zealand white rabbits, weighing approximately

1–1.5 kg were obtained from Laboratory Animal Resource

Section of IVRI, Izatnagar. They were maintained in

disinfected cages of small animal house of the Division of

Parasitology and were fed ad libitum. Rabbits were used for

rearing of Theileria annulata free H. a. anatolicum and also

to raise specific antibodies. The experimental animals were

maintained as per the approved guidelines laid down by

the committee for the purpose of control and supervision

of experimentation on animals, a statutory Indian body.

Laboratory rearing of Theileria annulata infection free

Hyalomma anatolicum anatolicum

The H. a. anatolicum Izatnagar isolate was maintained in

the Entomology Laboratory of the Division of Parasitol-

ogy for the last 15 years. Briefly, healthy New Zealand

white rabbits of 1 year old and 1.5–2 kg in weight were

used for feeding of ticks. To avoid stress on animals, 6–8

rabbits were maintained simultaneously. Normally, two

rabbits were utilized for each feeding cycle. After each

feeding cycle, the animals were kept free for at last

20 days to 1 month.

For feeding on large animals, cross-bred male calves of

4–8 months old were maintained from the initial stage

of birth in the tick and fly proof shed of the Division of

Parasitology. The animals were examined regularly to

maintain the disease free condition. The T. annulata-free

status of the calves was ascertained by periodical exami-

nation of Giemsa stained blood smears (Ghosh and

Azhahianambi, 2007). All the off the host ticks were

maintained in desiccators which were kept at 28�C in

incubator. Except when the light was switched on for

observation, the interior of the incubator was constantly

kept dark. The humidity was maintained by keeping a

10% solution of KOH at the base of the desiccators

(Solomon, 1951). The condition provided a relative

humidity between 85% at 28�C temperature.

Reverse transcription-polymerase chain reaction

amplification of Haa86 gene, cloning and sequencing

Total RNA from the eggs of H. a. anatolicum was isolated

by standard method. Briefly, 35 mg of matured fresh eggs

were treated with liquid nitrogen for 5 min. Immediately,

the frozen eggs were used for total RNA isolation using

RNeasy total RNA isolation kit (Qiagen, GmbH, Hilden,

Germany). The amount of isolated RNA was measured

using Perkin Elmer Spectrophotometer and was stored at

)20�C using equal volume RNA storage buffer (Ambion).

The forward primer (HF2) was designed with BamH1

restriction site (HF2-5¢-CGGC GGATCC TTG TTC GTT

GGC GCT ATT TTG CTC AT-3¢) and the reverse primer

(HR2) was designed with 5¢-Kpn1 and Xba1 (HR2-5¢-CCC GGTACC TCTAGA TGC AAC GGA GGC GGC

CAG TAA CAG GA-3¢) for subsequent cloning of the

PCR product. The detail of cloning of targeted gene has

been elaborated in Azhahianambi et al. (2009). The PCR

product digested with BamH1 and Xba1 and was cloned

into a prokaryotic expression vector pPROEXHTb (Life

technologies, CA, USA) digested with BamH1 and Xba1.

The positive clones were screened out based on ampicillin

resistance. The plasmid with 1965 bp Haa86 gene (Bm86

orthologue of H. a. anatolicum) was designated as pPRO-

HA86F. Both the strands of the insert were sequenced by

the dideoxi chain termination method. The nucleotide

sequence (ORF) information of Bm86 homologue of

H. a. anatolicum Izatnagar isolate (Genbank accession no.

EU665682) and its deduced amino acid sequence were

aligned with the existing sequence information viz., Bm86

orthologue of H. a. anatolicum Ludhiana isolate (origi-

nated from Punjab state) (Genbank accession no.

AF347079), Bm86 of Rhipicephalus (Boophilus) microplus

(Yeerongpilly, Australia) (Genbank accession no.

L. Jeyabal et al. Efficacy of recombinant tick protein Haa86

ª 2010 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 57 (2010) 96–102 97

M29321) and Bm95 of Bm86 resistant R. (B.) microplus

Argentinean strain (Genbank accession no. AF150891)

using GeneTool.

Removal of signal sequence and C-terminal anchoring

sequence by PCR and expression study

For expression of the gene in prokaryotic system, 144 bp

from 5¢-end and 96 bp from 3¢-end was deleted from the

ORF of Haa86 by PCR as described by Azhahianambi et al.

(2009). The shortened gene was further cloned in expres-

sion vector pET32a (Novagen, Merck Bio Sciences, Merck

KGaA, Darmstadt, Germany). The pET Trx fusion system

32 (Merck Bio Sciences) is designed for cloning and high

level expression of polypeptide sequences fused with the

109 amino acid Trxtag� (Merck Bio Sciences) thioredoxin

protein. Many proteins which are insoluble in Escherichia

coli tend to become soluble when fused with TrxTag

sequence. The thioredoxin reductase mutation has been

shown to allow the formation of disulphide bonds in the

E. coli (BL21) system. The presence of His Tag sequence

facilitates protein purification. For cloning the PCR prod-

uct and the vector pET32a were digested with EcoR1 and

Xho1. The digested DNA were ligated, transformed and

cloned in E. coli BL21pLys (Novagen). The recombinant or

genetically modified E. coli BL21pLys was selected based on

chloramphenicol and ampicillin resistance. Four to five

clones termed as E. coli–pETHA86 were selected for the

expression study. The clones were grown for 3–4 h in the

presence of chloramphenicol (34 lg/ml) and ampicillin

(100 lg/ml) and were induced with 1 mm isopropyl-beta-

thiogalactopyranoside and were grown further for 6–7 h.

The samples were spun at 10 000 g for 10 min and the

supernatants were resolved in 8% SDS-PAGE along with

protein molecular weight marker (Bangalore Genei,

Bangalore, India). The expressed protein was purified by

Ni-NTA affinity chromatography (Qiagen). The immuno-

genic property of the expressed protein (rHaa86) was char-

acterized by western blotting using anti-Bm86 antibodies

raised in rabbits (Azhahianambi et al., 2009). The appro-

priate concentration of recombinant Ha86 (rHa86) was

resolved in 8% SDS-PAGE under reducing condition and

transferred to the poly(vinylidene fluoride) membrane.

The membrane strips were incubated with rabbit anti-

Bm86 antibody 1 : 150 dilution in 1% skimmed milk in

phosphate-buffered solution (PBS)-containing 0.5% Tween

20 (PBST) for 2 h at room temperature. After washing five

times in PBST the strips were incubated in goat anti-rabbit

IgG-ALP (Sigma, St Louis, MO, USA) 1 : 1500 dilution in

1% skimmed milk in PBST for 2 h at room temperature.

The membrane strips were washed again with PBST and

subsequently incubated in the substrate solution [10 ml of

alkaline PBS (pH 9.5) + 100 ll of nitro blue tetrazolium

stock solution + 100 ll of 5-bromo-4-chloro-3-inodoyl-

phoshate stock solution]. The reaction was stopped by

placing the membrane strips in distilled water.

Immunization and challenge of calves

Ten calves, aged approximately 7 months were divided

randomly into two groups comprising of five animals in

each group. The frozen rHaa86 was thawed and emulsified

thoroughly with equal volume of adjuvant (10% Monta-

nide 888 in mineral oil; SE PPIC, Paris, France). All the

animals of group 1 were inoculated with 400 lg of rHaa86

intramuscularly on day 0, 400 lg on day 30 and 100 lg

on day 60. The corresponding control animals were inocu-

lated with equal volume of adjuvant on the same day.

Adults ticks of H. a. anatolicum infected with T. annulata

were raised for challenge study along with non-infected

ticks. Two 4 months old cross bred calves were inoculated

subcutaneously with a stabilate of T. annulata equivalent

to two infected ticks (ground up H. a. anatolicum

infected with T. annulata). Uninfected nymphs of labora-

tory reared H. a. anatolicum were allowed to feed on the

infected calves when intra-erythrocytic piroplasms

were detected in the blood smear. The fully engorged

nymphs were collected, reared in the laboratory to obtain

T. annulata infected adults.

Each calf (groups 1 and 2) was challenged on day 97

post-immunization with 50 uninfected adults of both

sexes (male and females in 1 : 1 ratio) and eight infected

female adults of H. a. anatolicum. The following parame-

ters were recorded for evaluation of the effect of immu-

nity to ticks (Fragoso et al., 1998).

1 The number of engorged adult female ticks dropped

from each animal was recorded.

2 Dropped engorged ticks were weighed individually.

3 The engorged female ticks were incubated for laying

eggs and the egg masses were weighed.

4 DT% = 100 (1 ) NTV/NTC), where DT% is the per

cent reduction of females, NTV, the number of females

dropped from the animals of group1 and NTC, the num-

ber of females dropped from the animals of group 2.

5 DO% = 100 (1 ) PATV/PATC), where DO% is the

per cent reduction of mean weight of eggs, PATV the

mean weight of eggs of females fed on animals of group 1

and PATC the mean weight of eggs of females fed on

animals of group 2.

6 DR% = 100 (1 ) PMTV/PMTC), where DR% is the

per cent reduction of mean weight of adult females,

PMTV the mean weight of adult females dropped from

the animals of group 1, and PMTC the mean weight of

adult females dropped from the animals of group 2.

7 E% = 100 [1 ) (CRT · CRO)], Where E% is the effi-

cacy of antigen, CRT is the reduction in the number of

Efficacy of recombinant tick protein Haa86 L. Jeyabal et al.


adult females NTV/NTC, CRO is the reduction in egg lay-

ing capacity, PATV/PATC (PATV, the mean weight of eggs

of ticks fed on the animals of group 1)/PATC, the mean

weight of eggs of females fed on animals of group 2.

Enzyme-linked immunosorbent assay

The rHaa86 was eluted from 12% non-reducing polyacryl-

amide gels. The gel slices were mixed thoroughly with

PBS, pH 7.4 and after incubating the mixture over night at

4�C, the targeted protein was collected from the superna-

tant by centrifugation. The eluted protein was checked by

SDS-PAGE. Blood samples were collected aseptically from

all calves during pre- and post-tick challenge period in a

regular interval. Sera were separated, aliquoted and stored

at )20�C till further use. The humoral immune response

to rHaa86 antigen was monitored by indirect ELISA. Ini-

tially checkerboard titration was used to optimize the

reagents. After optimization, the eluted antigen was

applied to the microtire plate in a concentration of 4 lg/

ml. The collected sera were diluted into 1/50 and were

used in triplicate wells. Anti-bovine peroxidase conjugate

(Sigma Chemical Company, USA) was used at a dilution

of 1 : 10000 as secondary antibody. The reaction was

stopped with 50 ll of 3 n HCl per well, and absorbance

was recorded by microplate ELISA reader (Tecan-Sunrise,

Grodig, Austria), as the mean OD492 of triplicate samples.

Post-challenge clinical observations

Daily examination of the calves was performed following

challenge infestation. The level of enlargement of sub-

mandibular lymph nodes and rectal temperature were

recorded. Examination of stained biopsy smear of lymph

nodes was performed from day 4 post-challenge (PC) to

estimate piroplasm parasitaemia. The estimation of clini-

cal severity of theileriosis was based on the presence of

sustained fever combined with symptoms like inappetance

and dyspnoea. The time period to detect pyrexia and time

period to detect macroschizonts in the lymph gland of

the calves was recorded.

Haematological studies

The packed cell volume (PCV%), total leucocytes count

(TLC; WBC · 103/cm2) and differential leucocytes count

(%) were performed according to Sastry (2000).

Statistical analysis

Significant differences in mean values from immunized

and control group of animals was determined by student

t-test (Snedecor and Cochran, 1968).

Results

Cloning and expression of Haa86

Initially 1965-bp full-length gene was cloned in

pPROEXHTb. After deletion of 144 bp from 5¢-end and

96 bp from 3¢-end, the shortened gene of 1755 bp was

cloned in expression vector and resultant plasmid construct

was designated as pETHA86. The identity of nucleotide

sequence of Bm86 homologue of H. a. anatolicum (Ha86)

with Bm86 homologue of H. a. anatolicum Ludhiana iso-

late (Ha98), Bm86 gene of R. (B.) microplus (Yeerongpilly,

Australia) and Bm95 gene of Bm86 resistant B. microplus

Argentinean strain are 99.3%, 76.1% and 77.4% respec-

tively. The identity of deduced amino acid sequence of

Ha86 with Ha98, Bm86 gene of R.(B.) microplus (Australia)

and Bm95 gene of Bm86 resistant B. microplus Argentinean

strain are 98.3%, 62.6% and 63.8% respectively.

Following expression in E. coli, the expressed protein of

97 kDa was affinity purified and the size of the protein is

consistent with the expected molecular mass considering

that the expression vector produced a recombinant pro-

tein fused with a 19-kDa thioredoxin protein.

Clinical and haematological responses in calves

The calves of group 1 were clinically normal following

inoculations of rHaa86 antigen during the pre-challenge

period of 97 days. There was no significant difference in

the TLC and PCV values between the immunized and

control groups of calves during the pre-challenge period.

The differential count (%) of lymphocytes, neutrophils

and monocytes in the peripheral blood of the calves were

in the normal range (Table 2). All the 10 calves of groups

1 and 2 developed theileriosis after challenge infestation

(Table 1). Fever (rectal temperature £39.50 C) was

detected after a mean period of 7.2 days in calves of

group 1 and 5.8 days in group 2. Enlargement of sub-

mandibular lymph nodes was recorded in all calves either

simultaneously or 1–2 days after onset of fever. The intra-

leuccocytic macroschizonts (Koch’s blue bodies) were

detected in the enlarged lymph nodes after a mean period

of 8.2 days in claves of group 1 and 6.0 days in claves of

group 2. The mean maximum macroschizont indexes

were 7.8% and 10.6% in the calves of group 1 and group

2 respectively. Two calves (identification no. 351 and

354) died in group 1 while all five calves died in control

group. The rectal temperature became normal in the

calves which survived in group 1 on day 20 PC.

Haematological examination revealed fall in PCV,

reduction in TLC in all calves after challenge infection.

Marked increase in differential count of monocyte was

recorded in the calves of group 1 that survived the infec-

tion on day 30 PC (Table 2).



Antibody response in calves

In group 1 animals, a significantly high anti-Haa86 anti-

body responses in comparison with group 2 animals were

detected after first boosting and antibody responses

reached at peak on 124 days of first immunization (DFI)

(3.75 · control value, CV, P < 0.001). At the time of

challenge (97 DFI), a significant (P < 0.01) increase of

3.0 · CV in the antibody response of calves immunized

with rHaa86 was noted and the anti-Haa86 antibodies

interfered with the feeding and reproductive efficiency of

ticks fed on immunized animals (Table 3).

Feeding and reproductive performances of Hyalomma

anatolicum anatolicum

The details of results obtained are presented in Table 4.

Adults started feeding on all the animals within 48 h of

their release. After 120 h of challenge, 7.4 ± 1.9% of

engorged females dropped from the immunized group

(group 1) while 28.3 ± 5.0% dropped from the animals of

control group (group 2) and the difference in dropping

per cent was found statistically significant (P < 0.01). A

reduction of 49.6 mg in the weight of ticks fed on group 1

animals in comparison with the ticks fed on group 2

animals was noted and the data were found statistically

significant (P < 0.01). The dropped ticks were kept for

oviposition and a reduction of 68.1 mg (P < 0.05) in the

egg masses laid by the ticks fed on immunized animals

was noted in comparison with the ticks fed on group 2

animals. The direct effect of immunization (DT%) on the

Table 3. Mean ± SE antibody response in calves immunized (Gr.-1)

by rHaa86 and control calves (Gr.-2)

Day Gr.-1 Gr.-2 Level of significance (P-value)

0 0.2 ± 0.02 0.18 ± 0.02 –

30 0.22 ± 0.08 0.18 ± 0.02 NS

60 0.44 ± 0.04 0.22 ± 0.04 <0.01

95 0.6 ± 0.04 0.2 ± 0.04 <0.01

107 0.68 ± 0.02 0.22 ± 0.02 <0.001

110 0.72 ± 0.04 0.24 ± 0.02 <0.001

116 0.8 ± 0.04 0.22 ± 0.04 <0.001

124 0.9 ± 0.02 0.24 ± 0.04 <0.001

129 0.84 ± 0.02 0.26 ± 0.03 <0.001

Table 2. Differential count (%; range) of blood cells of experimental calves (Gr.-1: immunized animals) and control animals (Gr.-2)

Blood cells

Prechallenge Day 15 post-challenge (PC) Day 30 post-challenge

Gr.-1 Gr.-2 Gr.-I Gr.-2 Gr.-I Gr.-2a

Neutrophils 21–30 15–30 14–20 08–06 13–16 –

Lymphocytes 62–77 45–60 30–35 32–49 62–86 –

Monocytes 02–08 02–07 04–08 15–18 01–25 –

aAnimals died before day 30 post-challenge.

Table 1. Clinical reactions of animals (control and immunized with rHaa86) following challenged with Hyalomma anatolicum anatolicuma

Group Calf No.

Pyrexia

dayb

MS

dayc

Piroplasm

day4

Max

% MS

Max %

Piroplasm

Min.

WBCx103/ml

Min.

PCV (%) R D

1 Immunized group 351 7.0 8.0 11 15.0 37.0 2.4 17.0 – 22.0

330 8.0 8.0 11 4.0 17.0 3.9 21.0 19.0 –

338 7.0 8.0 11 5.0 25.0 3.4 19.0 20.0 –

354 7.0 9.0 10 10.0 10.0 1.9 27.0 – 12.0

323 7.0 8.0 11 5.0 25.0 4.3 20.0 19.0 –

Mean 7.2 8.2 10.8 7.8 22.8 3.18 20.8

2 Control group 344 5.0 6.0 10 15.0 15.0 5.9 37.0 – 11.0

340 6.0 6.0 11 7.0 10.0 5.6 29.0 – 12.0

355 5.0 6.0 11 10.0 70.0 2.0 21.0 – 23.0

352 7.0 7.0 8.0 9.0 35.0 2.8 33.0 – 16.0

329 6.0 5.0 9.0 12.0 19.0 2.3 26.0 – 11.0

Mean 5.8* 6.0** 9.8 10.6 28.8 3.72 29.20***

R, day post-challenge when rectal temperature <39.5�C; D, day post-challenge when died.

*P < 0.01; **P < 0.01; ***P < 0.05.aUninfected and T. annulata infected adults of H. a. anatolicum.bFirst day rectal temperature ‡39.5ºC.cFirst day macroschizont detected in lymphnode biopsy.dFirst day piroplasms detected in erythrocytes.



number of female was 73.8%. The other entomological

parameters DO%, DR% and E% were calculated as 31.3%,

15.8% and 82.3% respectively.

Discussion

The recombinant antigen rHaa86 is expressed in E. coli

and process of expression and purification of the antigen

was standardized. Previously, the same gene was expressed

in yeast system, Pichia pastoris as a 120–140 kDa glycosy-

lated protein. The purification protocol was very lengthy

and the recovery of the expressed protein was compara-

tively low in comparison with the presently described

E. coli-expressed protein.

Following immunization of animals with the recombi-

nant antigen more number of engorged adult ticks were

dropped from the calves of group 2 (28.3 ± 5.0) if com-

pared with that dropped from calves of group 1

(7.4 ± 1.9). Further, the results indicated that there was a

significant reduction in the mean weights of engorged

adult and in the egg masses laid by the ticks fed on

immunized calves in comparison with those ticks

dropped from the control calves. The observations were

suggestive of a derangement in the feeding success in the

ticks fed on the calves of group 1. The result supported

the earlier observations of Ghosh et al. (1999) and Das

et al. (2000) who reported significant differences in the

feeding per cent of ticks fed on animals immunized by

purified larval antigen and on control animals. However,

none of these antigens were tested as recombinant

proteins.

There was a strong indication of contribution of tick

antigen (Haa86) in modulating the immune response of

the host to not only against the challenge infestation by

ticks but also to T. annulata infection as three calves in

group 1 survived the lethal challenge infection. A sus-

tained significantly high antibody response (P < 0.001) in

the calves of group 1 was indicative of development of a

protective humoral response in the calves against ticks as

had been proven conclusively (Brossard and Girardin,

1979; Labuda et al., 2006).

Successful transmission of Theileria sp. depends on the

sufficient doses of sporozoites in the saliva of the feeding

ticks. The sporogony in the unfed adult tick 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 because

of host’s immunity was reflected by the reductions in the

dropping rate, engorgement weight and egg masses in the

ticks fed on calves of groups 1 (Table 4). These events

were expected to cause inhibited maturation of sporozo-

ites and subsequent introduction of lesser quantum of

T. annulata infection by the challenged ticks to immu-

nized calves. The observations were in agreement with a

previous report of Fivaz et al. (1989). Moreover, a mini-

mum of 72 h of tick attachment is reported essential

(Singh et al., 1979) for active transmission of T. annulata.

It was observed that the animals which rejected more

number of ticks withstood the challenge thus confirming

the effect of anti-Haa86 antibodies at the initial stage of

feeding.

The present experiment is the first immunization and

challenge trial using the E. coli-expressed recombinant

antigen of H. a. anatolicum, rHaa86. Besides, the direct

effect on tick stages, the effect of immunization on limit-

ing transmission of T. annulata is also evaluated. An

increase in rejection per cent and decrease in reproductive

index of ticks fed on group 1 animals and significantly

high DT% and E% of rHaa86 are comparable with Bm86

(GAVAC) vaccine. The E% of the Gavac vaccine against

larvae of different strains of B. microplus varied from 51%

to 91%. In the present study, 82.3% efficacy was obtained

by challenging the immunized calves with adults of H. a.

anatolicum. The E% obtained in the present study is fall-

ing within the range of the work reported by Canales

et al. (1997). The DT% of the Gavac vaccine against

different strains of B. microplus was 9–74%. Amongst the

10 strains, DT% of above 50% was recorded against two

strains. The DT% (73.8%) obtained in the present study

was comparable with the higher than the DT% obtained

using Gavac vaccine (Canales et al., 1997).

The animals were challenged with lethal dose of

T. annulata. The calves which survived showed initial

symptoms of the disease but recovered quickly and all the

haematological parameters reached at base level thereafter.

In this circumstance, it is expected that the severity of

theileriosis could be further reduced in case of a sublethal

challenge which is more common in actual field condi-

tions. The experiment has given a significant clue towards

the development of rHaa86 based vaccine against both

H. a. anatolicum and T. annulata.

Table 4. Feeding and reproductive performances of adults of Hya-

lomma anatolicum anatolicum released on immunized and control

calves (values are expressed as mean ± SE)

Groups

Mean ± SE

No. of

ticks

dropped

Wt. of

engorged

adults (mg)

Egg

masses (mg)

1 Immunized animals 7.4 ± 1.9* 263.2 ± 10.9* 149.3 ± 13.0**

2 Control animals 28.3 ± 5.0 312.8 ± 9.3 217.4 ± 19.5

DT% = 73.8; DR% = 15.8; DO% = 31.3; E% = 82.3.

*P < 0.01; **P < 0.05.



Acknowledgements

Authors thank Department of Biotechnology and Indian

Council of Agricultural Research, Government of India

for providing financial assistance to execute the technical

programme. 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 Coordina-

tion 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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Efficacy of rHaa86, an Orthologue of Bm86, Against Challenge Infestations of Hyalomma anatolicum anatolicum