Vaccine 32 (2014) 34883494

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Vaccine
j our nal home page: www. el sevi er . com/ l ocat e/ vacci ne
Subolesin: A candidate vaccine antigen for the control of cattle tick
infestations in Indian situation
Mukesh Shakya
a
, Binod Kumar
a,1
, Gaurav Nagar
a
, Jos de la Fuente
b,c
, Srikanta Ghosh
a,
a
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243122, India
b
SaBio, Instituto de Investigacin en Recursos Cinegticos IREC-CSIC-UCLM-JCCM, Ronda de Toledo s/n, 13005 Ciudad Real, Spain
c
Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA
a r t i c l e i n f o
Article history:
Received 1 February 2014
Received in revised form11 April 2014
Accepted 17 April 2014
Available online 30 April 2014
Keywords:
Anti-tick vaccine
Subolesin
Rhipicephalus (Boophilus) microplus
a b s t r a c t
Identication of cross-protective tick vaccine antigens is a challenging area of veterinary research. To
address this challenge, a recently identied candidate tick protective antigen, Subolesin (SUB), was tar-
geted in this research. The conservation of subolesin ortholog of Hyalomma anatolicum and Rhipicephalus
(Boophilus) microplus across different Indian strains was 98.199.4% (within species), while at the amino
acid level SUB sequence homology was 53.2% (between tick species). Recombinant R. (B.) microplus SUB
(rBmSu) was produced in Escherichia coli and characterized. Cross-bred cattle male calves (N =10) were
immunized with three doses of 100 g each of the rBmSu emulsied in 10% Montanide 888 at monthly
intervals on days 0, 30 and 60. The control group was injected with PBS in 10% Montanide 888. For the rst
tick challenge, calves were infested with larvae of R. (B.) microplus generated from 100 mg eggs 2 weeks
after last immunization (day 75). The immunization resulted in 16.3%, 8.0%, 9.4%, and 26.1% reduction
in female tick numbers (DT), weight (DW), oviposition (DO) and egg fertility (DF), respectively, when
compared to controls. In the subsequent challenge on day 105, DT, DW, DO and DF were reduced by
9.0%, 4.1%, 8.6%, and 24.2%, respectively, when compared to controls. The vaccine efcacy (E) was equal
to 44.0% and 37.2% after the rst and second challenges, respectively. The results showed a positive cor-
relation between antibody titers for both total IgG and IgG1 and E in the second but not in the rst tick
challenge. These results suggested the possibility of developing a SUB-based vaccine for control of cattle
tick infestations under Indian conditions.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
On a global basis, ticks are second to mosquitoes as vector of
pathogens causing disease to humans and animals [1]. Moreover,
ticks can cause severe toxic conditions leading to irritation, allergy
and paralysis in the host. Ticks and tick-borne diseases (TTBDs) are
ranked high in terms of their impact on livelihood of resource poor
farming communities in developing countries such as India [2]. It is
estimated that about 80% of the world cattle population are at risk
of TTBDs which can affect livestock industry signicantly [1,35].
Globally, the losses incurredby livestock industry due toTTBDs was
estimated in the range of 14,00018,000 million US$/year [6]. In
India, the annual cost of TTBDs control in cattle has been estimated

Corresponding author. Tel.: +91 9410261029.

E-mail addresses: sghoshtick@gmail.com, sghoshp@yahoo.co.in (S. Ghosh).
1
Present address: Department of Veterinary Parasitology, College of Veterinary
Science andAnimal Husbandry, JunagadhAgricultural University, Junagadh362001,
India.
in 498.7 million US$/year [2]. The immunological control of tick
infestations is one of the promising options to manage the grow-
ing problems associated with acaricide resistance, environmental
contamination and contamination of meat and milk products with
pesticide residues [7]. The limited success of the only commercially
available recombinant BM86proteinbasedtick vaccines, TickGARD
and Gavac is due to variable efcacy against different strains of R.
(B.) microplus, which may be linked to protein variability and/or
tick physiological factors [811]. Recently, Subolesin (SUB) was
identied as a candidate tick protective antigen [1214]. It is the
structural and functional ortholog of insect and vertebrate Akirin
and functions as a transcription factor in regulation of gene expres-
sion, thus affecting multiple cellular processes such as tick innate
immune response, feeding, reproduction and development [15].
Immunizationtrials usingrecombinant tickSUBdemonstratedpro-
tective efcacy against tick infestations, reduced vectorial capacity
of ticks [1618] and fertility of several arthropod vector species
[1921]. Moreover, Carreonet al. [22] recorded 83%efcacy against
tick infestations following immunization of White-tailed deer with
SUB. The results of these experiments suggested that the antigen
http://dx.doi.org/10.1016/j.vaccine.2014.04.053
0264-410X/ 2014 Elsevier Ltd. All rights reserved.
M. Shakya et al. / Vaccine 32 (2014) 34883494 3489
could be used for the development of a cross-protective vaccine
against different tick species. Under Indian conditions, a SUB-based
vaccine can be efcacious for control of tick infestations if the gene
is conserved in different strains of the major cattle tick species,
Rhipicephalus (B.) microplus and Hyalomma anatolicum.
The objectives of this research were to characterize SUB
orthologs in different geographic strains of R. (B.) microplus and H.
anatolicumand the production of recombinant R. (B.) microplus SUB
(rBmSu) for the characterization of its protective efcacy against
cattle tick infestations.
2. Material and methods
2.1. Animals
Cross-bred cattle male calves (Bos taurus male Bos indicus
female), (n=10), and healthy NewZealand white rabbits were used
in the study. The experimental animals were maintained as per the
approvedguidelines laiddownby the Committee for the Purpose of
Control and Supervision of Experimentation on Animals (CPCSEA),
a statutory Indian body.
2.2. Tick colonies
The homogenous acaricide susceptible R. (B.) microplus [Indian
Veterinary Research Institute (IVRI) line I; national registra-
tion no. NBAII/BM/1/1998] and H. anatolicum (IVRI line II;
NBAII/IVRI/HA/1/1998) were used for RNA isolation and the R. (B.)
microplus IVRI line I was used for the subsequent challenge study.
2.3. Tick strains
The different strains of R. (B.) microplus and H. anatolicumwere
collected following two steps stratied sampling procedures. The
collected ticks were washed thoroughly, classied, weighed indi-
vidually, labeled and stored at 80

C.
2.4. RNA extraction, RT-PCR, cloning and sequencing of subolesin
gene
Total RNA was isolated from adults of tick reference colonies
and different eld strains using Trizol (Life Technologies, Carls-
bad, CA, USA). Reverse transcription was performed using the rst
strand cDNA synthesis kit (Thermo Scientic, USA). PCR amplica-
tion of the subolesin gene was standardized using different primer
sets designed from the conserved regions of subolesin orthologs
in different tick species [14]. The PCR products were cloned using
the InsTAclone
TM
PCR cloning Kit (Thermo Scientic, USA) and PCR
positive clones were sequenced. The internal primer set (HSF and
HSR; Supplementary Table S1) was designed fromthe rst identi-
ed H. anatolicumsubolesin ortholog sequence and used to identify
PCR-positive clones. At least three different clones from each PCR
product were sequenced to rule out any sequencing errors.
Supplementary Table S1 can be found, in the online version, at
http://dx.doi.org/10.1016/j.vaccine.2014.04.053.
2.5. Sequence analysis
Nucleotide sequence alignment was performed using BLAST
(NCBI). All nucleotide and their deduced amino acid sequences
were analyzed using the program MegAlign (DNAstar, USA). The
deduced amino acid sequences were used to search for orthologs in
the non-redundant GenBank protein sequence database by BLASTP
analysis. The retrieved sequences of hard ticks and dipteran were
alignedusing ClustalWandthe phylogenetic tree constructedusing
the neighbor-joining method in Mega4 package [23]. Dipteran
sequences were used as outgroups. Gaps were treated as pairwise
deletions and amino acid distances were calculated using the Pois-
son model and branch supports were estimated using bootstraps
analysis (10,000 bootstraps).
2.6. Expression of recombinant SUB
The predicted mature protein coding sequence for SUB was
expressed in Escherichia coli BL21(DE3) pLysS (Novagen, USA)
using the expression vector pET32(a) (Novagen). For unidirectional
cloning, forward primer RmS-F and reverse primers RmS-R were
designed having restriction sites for EcoRI and HindIII at forward
and reverse primer, respectively. The PCRproducts were subcloned
into the pET32(a) vector digested with EcoRI and HindIII. Recombi-
nant plasmids were used to transform E. coli-Novablue (Novagen)
and positive clones were selected by PCR. The plasmid of positive
clones was isolated and transformed into E. coli BL21(DE3) pLysS.
For expression, 10 transformed colonies were grown individually
at 37

C in 10ml LB broth medium(Himedia, India) supplemented

with ampicillin and chloramphenicol. The recombinant SUB syn-
thesis was induced with 1mMIPTG and the culture was grown for
anadditional 5hat 37

C. The expressionwas checkedby SDS-PAGE

and the colonies showing good production of recombinant protein
were selectedfor scale upproductionandpurication. The selected
clones were grown and induced as above and centrifuged. The cell
pellet was resuspended in lysis buffer (8Murea, 100mMNaH
2
PO
4
,
10mM Tris and 5mM imidazole, pH8.0), incubated at 22

C for 1h,
sonicated and centrifuged. The supernatants were mixed with Ni-
NTA resin (Qiagen, Germany), loaded into small plastic columns
and the recombinant SUB (rBmSu) was eluted, dialyzed and con-
centrated using cut-off device (Pall lter, UK) and stored at 20

C
in the presence of a protease inhibitor cocktail.
2.7. Western blot
The extracts from partially fed adults of R. (B.) microplus was
prepared as described previously [24]. Rabbits were immunized by
subcutaneous injection of 1mg (total dose) of tick extracts emul-
sied with Freunds complete adjuvant (Pierce, USA) followed by
two booster doses 2 weeks apart by the same route using Freunds
incomplete adjuvant (Pierce). Rabbits were bled directly from the
heart 1 week after the last immunization and sera were isolated
and stored at 20

C.
For Westernblot analysis, therBmSuwas resolvedbySDS-PAGE,
transferred to the PVDF membrane and probed sequentially with
hyperimmune rabbit sera raised against tick extracts, secondary
antibodies and chromogenic substance, DAB (diaminobenzidine).
The reaction was stopped by washing the membrane strips in dis-
tilled water.
2.8. Immunization trial
Ten animals, aged 68 months were divided randomly into two
groups comprising six animals in group 1 and four animals in group
2. The rBmSu was emulsied thoroughly with equal volume of 10%
Montanide 888 in mineral oil. The nal concentration of rBmSu in
vaccine preparation was maintained at 50g/ml. The immuniza-
tions werecarriedout ingroup1animals with2ml (100g)/animal
by deep intramuscular inoculation in the glutial muscle and neck at
0, 30 and 60 days. Group 2 animals were considered as controls and
inoculated with sterile PBS in 10% Montanide 888 in mineral oil. All
the animals were checkedregularly for any signs of local reactionor
clinical abnormalities post immunization. Blood samples were col-
lected from each animal before rst immunization and at 15 days
3490 M. Shakya et al. / Vaccine 32 (2014) 34883494
Fig. 1. PCR amplication and analysis of subolesin gene. (A and B) Subolesin ortholog of Hyalomma anatolicum (474bp) and Rhipicephalus (Boophilus) microplus (486bp),
respectively. 100bp plus DNA ladder from Thermo Scientic (USA). (C and D) Alignment of deduce amino acid sequences of subolesin gene of different isolates of R. (B.)
microplus and H. anatolicum, respectively. The amino acid substitution fromdeduce amino acid sequences are highlighted. (E) Neighbor-joining analysis of tick and dipterans
Subolesin proteins. The evolutionary distances were computed using the Poisson correction method. Branch support value (10,000 bootstraps) for nodes are indicated.
intervals after primary immunization till day 120 and the serum
samples were collected and stored at 20

C.
2.9. Characterization of the antibody response in immunized
animals
The three variables i.e., concentration of antigen, dilution of
primary and secondary antibodies were optimized employing the
checkerboard method [25]. The optimized conditions included
antigen concentration for coating, 2g/ml, dilution of sera 1:200
for IgG, 1:400 for IgG1 and 1:800 for IgG2, dilution of sec-
ondary antibodies 1:2000 for IgG, 1:8000 for IgG1 and 1:12000
for IgG2. Microtiter plates were coated with 100l antigen/well
overnight and the wells were blocked using 5% skimmed milk.
Pre-immunization cattle sera were used as negative controls.
Each diluted serum was added in triplicate wells, incubated at
37

C for 2h, washed and the optimized dilution of rabbit anti

bovine IgG-HRP, IgG1-HRP and IgG2-HRP conjugates were added
in each well, incubated and washed. The peroxidase mediated
color was developed for 20min at room temperature using o-
phenylenediamine dihydrochloride (Pierce, USA) in citrate buffer,
pH 5.0. The optical density (OD) was read at 492nm in an
ELISA reader (Tecan Sunrise, Austria). Antibody titers between
vaccinated and control cattle were compared by ANOVA test
(P=0.05).
2.10. Challenge infestation and data analysis
Fifteen days after the last immunization (day 75), each animal
was challengedwith68days oldR. (B.) microplus larvae emanating
from100mg of eggs. Animals were again challenged on day 120 to
assess the duration of protective effect of the immunization. Ani-
mals were challenged as previously described [26]. Adult engorged
female ticks dropped fromcattle were collected daily, counted and
weighed. The effect of immunization on tick infestations was eval-
uated employing the formulae:
Effect onthe number of adult female ticks (DT)
= 100

NTV
NTC

,
where, NTV is the number of adult female ticks dropped fromvac-
cinated group and NTC is the number of adult female ticks dropped
fromthe control group.
Effect ontickweight (DW) = 100

WTV
WTC

where WTV is the average adult female tick weight in the vacci-
nated group and WTC is the average adult female tick weight in the
control group.
Effect onoviposition(DO) = 100

PATV
PATC

M. Shakya et al. / Vaccine 32 (2014) 34883494 3491

Fig. 2. (A) The percentage of sequence identity among IVRI line I, IVRI line II and the Subolesin fromother ticks and dipterans. (B) Alignment of ticks and dipteran Subolesin
proteins. The amino acid substitution fromdeduce amino acid sequences are highlighted. Accession numbers in GenBank are as follows: Ornithodoros erraticus (ADN66054.1),
O. (ADN66053.1), Rhipicephalus appendiculatus (ABA62331.1), R. (B.) microplus (ABZ89745.1), R. sanguineus (ABA62332.1), Dermacentor variabilis (AAV67034.2), D. marginatus
(ABA62333.1), Ixodes scapularis (XP002414493.1), I. ricinus (ABA62325.1), Amblyomma hebraeum (ABY84524.1), A. americanum (ABA62326.1), Haemaphysalis longicornis
(ACA84004.1), H. qinghaiensis (ACA09713.1), Hyalomma marginatum(ABA62335.1), Aedes albopictus (ACF49499.1) and Glossina morsitans morsitans (ADD20629.1).
where PATV is the average egg masses laid by the ticks dropped
fromthe vaccinated group and PATC is the average egg masses laid
by the ticks dropped fromcontrol group.
Effect oneggfertility(DF) =
Eggweight
Engorgedlarvae weight
CRF % = Reductioninfertility

PPLOV
PPLOC

where PPLOV is the average weight of larvae per gram of eggs in

the immunized group and PPLOC is the average weight of larvae
per gramof eggs in the control group.
Vaccine efcacy(E) = 100 (1 (CRT CROCRF))
where CRT, CRO and CRF are the reduction in number of adult
females, oviposition and egg fertility of ticks fed on immunized
group of animals as compared to the control group.
3. Results
3.1. Cloning and sequencing
The subolesin gene of R. (B.) microplus and H. anatolicum was
amplied using the common forward primer (SuF1) and differ-
ent reverse primers (SuR2 for R. (B.) microplus and SuR1 for H.
anatolicum; SupplementaryTableS1) without anynonspecic reac-
tion. The complete coding region of the R. (B.) microplus subolesin
gene was amplied as a 486bp product and the same gene of
H. anatolicum was amplied as a 474bp product (Fig. 1A and B).
The internal primer set amplied a 270bp fragment of H. ana-
tolicum subolesin ortholog. The subolesin gene sequences from
different tick strains were submitted to GenBank (NCBI) with the
accession numbers: JQ713774JQ713787, JQ922396JQ922400,
JX431493JX431509.
3.2. Sequence analysis
The BLASTP analysis of the non-redundant GenBank protein
databaseusingtheSUBsequenceof either R. (B.) microplus or H. ana-
tolicumretrieved the same proteins. Alignments of deduced amino
acid sequences of SUB fromdifferent strains of R. (B.) microplus and
H. anatolicum are shown in Fig. 1C and D. The subolesin sequence
identity among different strains was 97.599.4%. The phylogenetic
analysis of the SUB amino acid sequences showed that the H. ana-
tolicum IVRI line II sequence is forming a separate clade while the
R. (B.) microplus IVRI line I sequence is coming within the clade of
hard ticks close to other strains of R. (B.) microplus (Fig. 1E).
When the deduced amino acid sequences of IVRI line I
(AFH57334) and IVRI line II (JX431493) were aligned together with
other ticks and dipteran SUB protein sequences, IVRI line II SUB
showed 53.2% identity with that of IVRI line I, between 48.2% and
3492 M. Shakya et al. / Vaccine 32 (2014) 34883494
Fig. 3. Expression and immunological characterization of SUB proteins. (A) Coomassie Brilliant Blue SDS-PAGE analysis of afnity puried recombinant SUB ortholog of R.
microplus (rBmSu); M protein molecular weight marker [Thermo Scientic, USA (Cat. No. Sm0431)]. (B)Western blot analysis of recombinant rBmSu probed with rabbit
anti-Boophilus antibodies, CS control sera, M protein molecular weight marker [Thermo Scientic, USA (Cat. No. Sm0431)]. (C) Mean humoral immune response for IgG,
IgG1 and IgG2 in calves of rBmSu immunized and control groups. (D and E) Correlation between antibody titer and vaccine efcacy during 1st challenge and 2nd challenge,
respectively.
57.0% identity with that of other hard ticks, and 40.542.4% iden-
tity with that of dipterans (Fig. 2A). The percent identity of IVRI
line I SUB protein with that of hard ticks was 77.4100%. Some SUB
regions were highly conserved amongst tick and dipteran species
(Fig. 2B).
3.3. Expression, purication and Western blot analysis of rBmSu
The rBmSu resolved at 38kDa on SDS-PAGE which is consistent
with the expected molecular size considering that the expression
vector produced a recombinant protein fused with a 20kDa thiore-
doxinproteintag(Fig. 3A). Followingprobingof thepuriedprotein
with anti-tick sera raised in rabbits, a strong reaction was detected
at 38kDa, while no reaction was observed against control sera
(Fig. 3B).
3.4. Immune response
In the rBmSu-immunized group, the serum IgG levels rose
to 26.6-fold of the pre-immunization value on day 30 post-
immunization (dpi), reached at 106.6-fold on 75th dpi and then
gradually decreased till 120th dpi but the value was 45.5-fold
higher than the pre-immunization value until the end of the
experiment (Fig. 3C). The IgG1 levels rst rose to 69.8-fold of
the pre-immunization value on 30th dpi, peaked at 257.8-fold of
the pre-immunization value on 75th dpi and then decreased but
remained higher than the pre-immunization value throughout the
experiment (Fig. 3C). The IgG2 levels rose to 24.5-fold of the pre-
immunization value on 30th dpi and peaked at 127.1-fold of the
pre-immunization value on 75th dpi and then gradually decreased
on 90th dpi onwards (Fig. 3C). Comparing the immune response
of individual animal it was observed that the IgG1 response was
strongly stimulated following immunization (Fig. 3C). Control
animals did not show anti-SUB antibodies at any time during the
experiment (Fig. 3C).
3.5. Effect of immunization on tick infestations
All the calves were clinically normal following immunization
and local reactions were not observed at the site of inoculation. The
unfed larvae started feeding within 48h of infestation. Engorged
female adults started to drop from the animals after 1819 days
of feeding. No signicant differences in the number of dropped
females were recorded amongst the groups. Comparing results
between immunized and control groups after the rst challenge,
signicant differences (P<0.001) were observed only in the DF
(Table 1). Following the 2nd challenge, signicant differences were
observed in the DO(P<0.01) and DF (P<0.001) (Table 1). The mean
vaccine efcacy for the two challenges was calculated as 40.65%.
3.6. Correlation between antibody response and vaccine efcacy
There was no signicant correlationbetweenantibody response
and E after the rst challenge as Pearson R values ranged from
0.212 to 0.045 and the variance in antibody response cannot be
explained by the variance in E (Fig. 3D). However, after the second
challenge 95.35% of the variance (R
2
=0.9535) in IgG1 was signi-
cantly (P<0.05) correlated with the E with the highest correlation
coefcient of 0.976. Similarly, 89.33% of the variance in IgG was
explained by the variance in E showing a correlation coefcient of
0.945 (Fig. 3E).
4. Discussion
The success story of Bm86-based vaccines [911] created
a lot of enthusiasm among the tick researchers to explore the
M. Shakya et al. / Vaccine 32 (2014) 34883494 3493
Table 1
Feeding and reproductive performance of adults of R. (B.) microplus fed on immunized and control calves.
Experimental group
a
R. (B.) microplus (susceptible, IVRI line)
Percent reduction, immunized/control
b
(meanSD)
DT DW DO DF E
Ist challenge immunized 11.7% 8.0% 9.4% 26.1
**
44.1%
120.0 149.0 78.4 0.6
44.0 156.0 74.4 0.9
102.0 128.0 56.5 0.6
105.0 152.0 78.1 0.6
94.0 124.0 60.0 0.6
(93 10) (141.86.2) (70.64.2) (0.670.08)
Control 62.0 154.0 81.1 0.9
125.0 164.0 80.4 0.9
128.0 150.0 74.2 0.8
105.0 149.0 78.5 0.9
135.0 154.0 80.0 0.9
(11013) (154.22.6) (78.01.5) (0.910.02)
2nd challenge immunized 9.0% 4.1% 8.6%
*
24.2 37.2%
363.0 113.4 45.5 0.4
243.0 103.4 45.6 0.5
412.0 107.3 44.5 0.5
340.0 116.9 49.2 0.5
240.0 100.0 47.5 0.5
(33119.0) (108.23.0) (46.60.7) (0.50.02)
Control 419.0 107.6 51.5 0.7
423.0 112.2 51.0 0.7
296.0 115.6 51.1 0.6
331.0 119.2 50.5 0.6
330.0 120.0 52.5 0.7
(364.432) (1153.0) (51.00.2) (0.660.01)
a
Cattle were randomly assigned to experimental groups (n=5), immunized and challenged with R. (B.) microplus larvae.
b
The percent reduction was calculated with respect to control group: DT, % reduction in tick infestations; DW, % reduction in tick weight; DO, % reduction in oviposition;
DF. % reduction in fertility. Following percent reduction the data for each cattle are shown and in parentheses the group average SD, for tick number, tick weight (mg),
oviposition (egg weight/tick weight, mg) and fertility (larvae weight/egg weight) and were compared by Students t-test with unequal variance between vaccinated and
control groups. Vaccine efcacy (E) was calculated as 100[1(CRTCROCRF)], where CRT, CROand CRF are the reduction in the number of adult female ticks, oviposition
and egg fertility as compared to the control group, respectively.
*
p<0.01.
**
p<0.001.
possibilities of identifying Bm86 homologues in different tick
species. Accordingly, the Bm86 homologue gene of H. anatolicum
was cloned, expressed and tested against experimental tick infes-
tations with homologous and heterologous strains with variable
efcacy [26,27]. Similarly, Bm86-immunized animals provided
partial protection against two prominent tick species of India [28].
Sequence divergence in vaccine antigens has been suggested as a
factor in the variable response to vaccination between tick species
and geographical strains [10,16]. Garcia-Garcia et al. [10] reported
that variations greater than 2.8% in the amino acid sequence of
the protein expressed would be sufcient to confer vaccination
inefciencies when recombinant antigens were used. Additionally,
it has beenshownthat tick physiological factors may also affect tick
vaccine efcacy [11]. The results of previous experiments clearly
suggested the need to have a better target for the development of a
tick vaccine cross-protective against different tick strains suitable
for Indian conditions. Tick SUB was shown to protect against
different tick species [18] and was thus selected as a candidate
protective antigen for vaccination against tick infestations in India.
As R. (B.) microplus and H. anatolicum are the two major tick
species infesting Indian cattle, the study of SUB orthologs in these
tick species is very important to develop an effective vaccine. In
the present study, the different strains of R. (B.) microplus and H.
anatolicum SUB orthologs showed very high sequence identity at
amino acid (97.599.4%) and nucleotide (99.2100%) levels within
the tick species. This result demonstrated that these genes are
highly conserved within tick species. However, sequence iden-
tity between R. (B.) microplus and H. anatolicum SUB (53.2%) was
lower than that reported between other tick species (57.098.2%)
[35]. Recently, immunization with mosquito Akirin has shown
protection against mosquito, tick and other arthropod vector
species [16,18,19,21,29,30,35]. The amino acid sequence homology
betweenA. albopictus mosquito SUBandsandies, Ixodes scapularis
and R. (B.) microplus SUB is 68.5%, 48.4% and 55.3%, respectively
[35]. These results suggested that although the H. anatolicum SUB
ortholog is forming a separate clade, the cross-protective potential
of rBMSu could not be ruled out.
In most of the previous experimental trials with recombinant
SUB, Montanide ISA 50V was used as adjuvant [3133,35]. In some
of these experiments, anti-SUB antibody titers increased after the
rst immunization and then decreased after the second immuniza-
tion possibly due to instability of the vaccine formulation [32]. In
another experiment, a high anti-SUB titer was recorded after the
rst immunization in calves using the same adjuvant and remained
signicantly higher throughout the experiment when compared
to adjuvant/saline-injected controls [31]. In this experiment, the
antibody titers at tick infestation time positively correlated with
the number of ticks collected after feeding [31]. In the experiment
reportedhere, antibodytiters increasedafter the last immunization
and positively correlated with E after the second tick challenge.
These results were similar to the results obtained earlier using
Bm86 [34] and Haa86 [26] antigens, also suggesting that the reduc-
tionincattle tick infestations were the result of anti-SUBantibodies
in vaccinated cattle.
The E% obtained here was similar to results reported in pre-
vious experiments using vaccination with tick SUB [18,21,22,33].
Interestingly, in the experiments reported here the most important
effect of SUB immunization was observed on tick DF, which has
been commonly found in previous experiments with SUB/Akirin
vaccines [21].
3494 M. Shakya et al. / Vaccine 32 (2014) 34883494
These results suggested that SUB could be used to develop
vaccines for the control of cattle tick infestations in India. The
high sequence homology between tick strains, the effect of immu-
nization on tick egg fertility and the positive correlation between
antibody titers in cattle and E suggested that vaccination with
rBmSucouldbe usedtoreduce tickpopulations andthus contribute
to the control of R. (B.) microplus tick strains infesting cattle inIndia.
Future experiments are needed to characterize the effect of rBmSu
on H. anatolicuminfestations and the efcacy of the vaccine under
eld conditions and in combination with other control measures.
Conict of interest
There is no conict of interest that could be perceived as preju-
dicing the impartiality of the research reported.
Acknowledgements
We thankDirector of IVRI for providingresearchfacilities. Senior
author is highly thankful to the Indian Council of Agriculture
Research(ICAR) for providing Senior Researchfellowship. The tech-
nical support provided by the Senior Research Fellow, Anil Kumar
Sharma and Sachin Kumar and laboratory staff, Naresh Kumar of
the entomology laboratory is highly acknowledged.
References
[1] Jongejan F, Uilenberg G. The global importance of ticks. Parasitol
2004;129:S314.
[2] MinjauwL, Mc Leod A. Tick borne diseases and poverty. The impact of tick and
tick borne diseases on the livelihoods of small-scale and marginal livestock
owners in India and eastern and southern Africa. In: Research report. DFIDani-
mal healthprogramme. UK: Centrefor Tropical VeterinaryMedicine, University
of Edinburgh; 2003.
[3] FAO/OIE/WHO. Animal health year book; 1994. p. 25.
[4] Biswas S. Role of veterinarians in the care and management during harvest of
skininlivestockspecies. In: Proceedings of National Seminar onLather Industry
in Todays perspective Kolkata, India, November 1415, 2003. p. 624.
[5] Jonsson NN. The productivity effects of cattle tick [Rhipicephalus (Boophilus)
microplus] infestation on cattle, with particular reference to Bos indicus cattle
and their crosses. Vet Parasitol 2006;88:7992.
[6] De Castro JJ. Sustainable tick and tick-borne diseases control in livestock
improvement in developing countries. Vet Parasitol 1997;71:7797.
[7] Willadsen P. Tick control: thoughts on a research agenda. Vet Parasitol
2006;138:1618.
[8] de la Fuente J, Garcia-Garcia JC, Gonzalez DM, Izquierdo G, Ochagavia ME.
Molecular analysis of Boophilus spp. (Acari: Ixodidae) tick strains. Vet Parasitol
2000;92:20922.
[9] Sossai S, Peconick AP, Sales-Junior PA, Marcelino FC, Vargas MI, Neves ES, et al.
Polymorphism of the Bm86 gene in South American strain of the cattle tick
Boophilus microplus. Exp Appl Acarol 2005;37:199214.
[10] Garcia-Garcia JEC, Gonzalez IL, Gonzalez DM, Valdes M, Mendez L, Lamberti J,
et al. Sequence variations in the Boophilus microplus Bm86 locus and implica-
tions for immunoprotection in cattle vaccinated with this antigen. Exp Appl
Acarol 1999;23:88395.
[11] Popara M, Villar M, Mateos-Hernndez L, Fernndez de Mera IG, Marina A, del
Valle M, et al. Lesser protein degradation machinery correlates with higher
BM86 tick vaccine efcacy in Rhipicephalus annulatus when compared to Rhipi-
cephalus microlpus. Vaccine 2013;31:472835.
[12] Almazan C, Kocan KM, Bergman DK, Garcia-Garcia JC, Blouin EF, de la Fuente J.
Identication of protective antigens for the control of Ixodes scapulris infesta-
tions using cDNAexpresion library immunization. Vaccine 2003;21:1492501.
[13] de la Fuente J, Almazan C, Blouin EF, Naranjo V, Kocan KM. RNA interfer-
ence screening in ticks for identication of protective antigens. Parasitol Res
2005;96:13741.
[14] de la Fuente J, Almazan C, Blas-Machado U, Mangold VNAJ, Blouin EF, Gor-
tazar C, et al. The tick protective antigen, 4D8, is a conserved protein involved
in modulation of tick blood ingestion and reproduction. Vaccine 2006;24:
408295.
[15] Naranjo N, Aylln N, Prez de la Lastra JM, Galindo RC, Kocan KM, Blouin EF,
et al. Reciprocal regulation of NF-kB (Relish) and Subolesin in the tick vector
Ixodes scapularis. PLoS ONE 2013;8:e65915.
[16] Canales M, Almazan C, Naranjo V, Jongejan F, de la Fuente J. Vaccination
with recombinant Boophilus annulatus Bm86 ortholog protein, Ba86, pro-
tects cattle against B. annulatus and B. microplus infestations. BMC Biotechnol
2009;9:2935.
[17] Prudencio CR, Perez de la Lastra JM, Canales M, Villar M, de la Fuente J. Map-
ping protective epitopes in the tick and mosquito Subolesin ortholog proteins.
Vaccine 2010;28:5398406.
[18] de la Fuente J, Moreno-Cid JA, Canales M, Villar M, Lastra JMP, Kocan KM, et al.
Targeting arthropod Subolesin/Akirin for the development of a universal vac-
cine for control of vector infestations and pathogen transmission. Vet Parasitol
2011;181:1722.
[19] Harrington D, Canales M, de la Fuente J, de Luna C, Robinson K, Guy J, et al.
Immunisation with recombinant proteins subolesin and Bm86 for the control
of Dermanyssus gallinae in poultry. Vaccine 2009;27:405663.
[20] Moreno-Cid JA, Jimenez M, Cornelie S, Moreno-Cid JA, Jimnez M, Cornelie S,
et al. Characterization of Aedes albopictus akirin for the control of mosquito and
sand y infestations. Vaccine 2011;29:7782.
[21] de la Fuente J, Moreno-CidJA, GalindoRC, AlmaznC, KocanKM, MerinoO, et al.
Subolesin/Akirinvaccines for thecontrol of arthropodvectors andvector-borne
pathogens. Transbound Emerg Dis 2013;60(Suppl. 2):1728.
[22] Carreon D, de la Lastra JM, Almazan C, Canales M, Ruiz-Fons F, Boadella M,
et al. Vaccination with BM86, subolesin and akirin protective antigens for the
control of tick infestations in white tailed deer and red deer. Vaccine 2012;30:
2739.
[23] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics
analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:15969.
[24] Ghosh S, Khan MH, Gupta SC. Immunization of rabbits against Hyalomma ana-
tolicumanatolicumusing homogenates fromunfed immature ticks. IndianJ Exp
Biol 1998;36:16770.
[25] Crowther JR. The ELISA guidebook. Methods in molecular biology, Vol. 149.
Humana Press; 2001, p-421.
[26] Kumar B, Azhahianambi P, Ray DD, Chaudhuri P, de la Fuente J, Kumar R,
et al. Comparative efcacy of rHaa86 and rBm86 against Hyalomma ana-
tolicum anatolicum and Rhipicephalus (Boophilus) microplus. Parasite Immunol
2012;34:297301.
[27] Azhahianambi P, De La Fuente J, Suryanarayana VV, Ghosh S. Cloning, expres-
sion and immunoprotective efcacy of rHaa86, the homologue of the Bm86
tick vaccine antigen, fromHyalomma anatolicumanatolicum. Parasite Immunol
2009;31:11122.
[28] Kumar B, Murugan K, Ray DD, Ghosh S. Efcacy of rBm86 against Rhipicephalus
(Boophilus) microplus (IVRI-I line) andHyalomma anatolicumanatolicum(IVRI-II
line) infestations on bovine calves. Parasitol Res 2012;11:62935.
[29] Canales M, NaranjoV, AlmazanC, MolinaR, TsurutaSA, SzaboMPJ. Conservation
and immunogenicity of the mosquito ortholog of the tick protective antigen,
subolesin. Parasitol Res 2009;105:97111.
[30] Canales M, Labruna MB, Soares JF, Prudencio CR, de la Fuente J. Protective
efcacy of bacterial membranes containing surface-exposed BM95 antigenic
peptides for the control of cattle tick infestations. Vaccine 2009;27:72448.
[31] Merino O, Almazan C, Canales M, Villar M, Moreno-Cid JA, Estrada-Pena A,
et al. Control of Rhipicephalus (Boophilus) microplus infestations by the combi-
nation of subolesin vaccination and tick autocidal control after subolesin gene
knockdown in ticks fed on cattle. Vaccine 2011;29:224854.
[32] Almazan C, Lagunes R, Villar M, Canales M, Rosario-Cruz R, Jongejan F, et al.
Identication and characterization of Rhipicephalus (Boophilus) microplus can-
didate protective antigens for the control of cattle tick infestations. Parasitol
Res 2010;106:4719.
[33] Almazan C, Moreno-Cantu O, Moreno-Cid JA, Galindo RC, Canales M, Villar M,
et al. Control of tick infestations in cattle vaccinated with bacterial membranes
containing surface-exposed tick protective antigens. Vaccine 2012;30:26572.
[34] de la Fuente J, Rodriguez M, Redondo M. Field studies and cost effectiveness
analysis of vaccinationwithGavac
TM
against the cattle tick Boophilus microplus.
Vaccine 1998;16:36673.
[35] Moreno-Cid JA, Prez de la Lastra JM, Villar M, Jimnez M, Pinal R, Estrada-Pe na
A, et al. Control of multiple arthropod vector infestations with subolesin/akirin
vaccines. Vaccine 2013;31:118796.