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d86 mRNA expression profile in Hyalomma scupense lifetages, could it contribute to explain anti-tick vaccine effectiscrepancy between adult and immature instars?
ourad Ben Saida, Yousr Galaïa,b, Melika Ben Ahmedb, Mohamed Gharbia,osé de la Fuentec,d, Mohamed Jedidia, Mohamed Aziz Darghoutha,∗
Laboratoire de Parasitologie, Ecole Nationale de Médecine Vétérinaire, 2020 Sidi Thabet, IRESA and La Manouba University, TunisiaLaboratoire d’Immunologie Clinique, Institut Pasteur de Tunis, TunisiaInstituto de Investigación en Recursos Cinegéticos IREC-CSIC-UCLM-JCCM, Ronda de Toledo s/n, 13005 Ciudad Real, SpainDepartment 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
rticle history:eceived 27 February 2013eceived in revised form 23 July 2013ccepted 24 July 2013
Bm86 midgut protein has been used in order to control ticks of the Hyalomma genus. Pre-vious studies demonstrated the inefficacity of this antigen in the control of Hyalommascupense, whereas recombinant Hd86 antigen, the Bm86 ortholog in H. scupense producedin Pichia pastoris, was protective against larval H. scupense tick stage infestations but ineffec-tive in the control of the adult stage. One possible explanation for this result is the variationin Hd86 expression levels between these two developmental stages. To test this hypothesis,Hd86 mRNA levels were characterized in H. scupense developmental stages. The expressionprofile of Hd86 demonstrated a significant variation between tick life stages and showeda significant reduction in the number of transcripts during feeding and, particularly aftermolting to adults. The most interesting result was noted after molting of engorged nymphsin unfed adults where the expression levels decreased significantly by 12.78 (10.77–17.39)(p < 0.001) and 9.25 (5.77–15.72)-fold (p < 0.001) in unfed males and unfed females, respec-tively. Comparing unfed nymphs to unfed adult ticks, the Hd86 expression levels decreasedby 13.82 (5.39–24.45) (p = 0.035) and 9.93 (2.87–22.08)-fold (p = 0.038) in males and females
respectively. Lower Hd86 mRNA levels in adult ticks should result in lower protein levelsand thus less antibody–antigen interactions necessary for vaccine efficacy in ticks fed onvaccinated animals. Thus, the observed differences in Hd86 expression profile betweenimmature and adult stages might explain, in part, the discrepancy of the Hd86 vaccineefficacy against these two life stages of H. scupense.
. Introduction
Please cite this article in press as: Ben Said, M., et al., Hd86 mRcould it contribute to explain anti-tick vaccine effect discrepan(2013), http://dx.doi.org/10.1016/j.vetpar.2013.07.035
The two host tick Hyalomma scupense (H. scupense) ishe natural vector of Theileria annulata the causative agentf tropical theileriosis (Bouattour et al., 1996, 1999). This
disease represents one of the most serious threats to thedevelopment of a purebred dairy cattle industry particu-larly for small stockholders who totalize together the majorpart of the Tunisian cattle herd (Gharbi et al., 2006).
In Tunisia, the prevention of tropical theileriosis is stillrelying on cattle treatment with pesticides which causes
NA expression profile in Hyalomma scupense life stages,cy between adult and immature instars? Vet. Parasitol.
environmental pollution (Darghouth et al., 1999; Mhadhbiet al., 2010) and resistance to these chemicals are alwaysreported (Graf et al., 2004; Willadsen, 2004; de la Fuenteet al., 2008). A T. annulata live attenuated vaccine was
Table 1Schedule of tick feeding employed for the synchronous feeding of severallife stages from H. scupense on rabbit.
Action Time point (days)
Placement of unfed larvae 0Collection of fed larvae 8Collection of unfed nymphs 11Collection of partially fed nymphs 17Placement of unfed males and females 0
Collection of partially fed males 3Collection of partially fed females 4
developed and assessed in Tunisia. However, its efficacyremains restricted to the prevention of clinical forms ofinfection (Darghouth et al., 2006; Gharbi et al., 2006;Darghouth, 2008).
Recently, an anti-tick vaccine strategy was developedagainst the Rhipicephalus microplus (R. microplus) tick vec-tor in order to reduce the cattle tick infestation and todecrease tick pathogens transmission (Willadsen et al.,1989, 1995; Graf et al., 2004). A tick gut protein (Bm86) iso-lated from R. microplus has shown its efficacy as an anti-tickvaccine against several tick species. Indeed, Bm86 vaccinesgive a high protection efficacy against R. annulatus infes-tations (Fragoso et al., 1998; Pipano et al., 2003; Canaleset al., 2009), partial cross-protection against Rhipicephalusdecoloratus, Hyalomma anatolicum, Hyalomma dromedariiand Rhipicephalus sanguineus (de Vos et al., 2001; Rodriguezet al., 2012; Odongo et al., 2007; Perez-Perez et al., 2010).However, this vaccine was demonstrated completely inef-fective against H. scupense infestation in cattle (Galaï et al.,2012) whilst the orthologous recombinant protein fromH. scupense rHd86 was found protective against imma-ture H. scupense tick stage infestation but ineffective in thecontrol of the adult tick stage (Galaï et al., 2012). Factorssuch as conformational epitopes, quantity of host antibod-ies ingested by the tick while feeding or intestinal antigenabundance might be of relevance in determining Hd86 vac-cine efficacy against H. scupense juvenile and adult stages.
To investigate the latter hypothesis, we have measuredin this work the Hd86 mRNA transcript levels throughoutthe H. scupense tick life stages by quantitative RT-PCR.
2. Materials and methods
2.1. Ticks and tick feeding
H. scupense ticks originating from farms located in thelocality of El Hessiene, area of Raoued (Governorate ofAriana, Tunisia) were maintained at the Laboratory of Par-asitology (ENMV) in order to obtain larvae. The previousgeneration of these ticks was used in an immunizationexperiment by Hd86 vaccine against larvae and adult stagesof H. scupense species. Free-living stages were kept at 20 ◦Cand 95% relative humidity. Ticks from unfed larvae of H.scupense were available at the start of the experiment. Atick free rabbit 3 months age was used to feed the ticks
Please cite this article in press as: Ben Said, M., et al., Hd86 mRcould it contribute to explain anti-tick vaccine effect discrepan(2013), http://dx.doi.org/10.1016/j.vetpar.2013.07.035
(Table 1). A circular patch covering the rabbit abdomenwas employed to release unfed larvae on the rabbit. Unfednymphs were collected from the rabbit before they werefixed again for blood meal. Engorged nymphs collected
PRESSitology xxx (2013) xxx– xxx
from the rabbit were allowed to molt at 28 ◦C and 95% rela-tive humidity. Mature unfed adults were allowed to partialfeeding on rabbit ears for three days prior to use (Table 1).
2.2. RNA isolation
Total RNA was isolated from 300 �l blood from rabbitprior to the tick feeding using the Trizol reagent liquid sam-ple (Invitrogen) according to the manufacturer’s protocol.
Tick RNA was isolated from different tick stages gener-ated from the same tick batch fed on the same rabbit inorder to minimize possible variations in tick gene expres-sion related to host or environment. Triplicate pools of,approximately, 100 mg of various life stages of H. scu-pense: unfed larvae, fed larvae, unfed nymphs, partially fednymphs, unfed males and females ticks and partially fedmales and females ticks were homogenized in 1 ml Tri-zol reagent (Invitrogen) using a pestle and liquid nitrogen.Then, samples were homogenized by a passage through24- and 27-gauge needles and centrifuged at 12,000 × gat 4 ◦C for 10 min to remove insoluble material and thesupernatant was frozen at −80 ◦C until RNA extraction.
Sample concentrations and purity were determinedwith a UV spectrophotometer at 260 nm (A260), 280 nm(A280) and 230 nm (A230) wavelengths. Only samples withA260/A280 and A260/A230 ratios between 1.8 and 2.2 wereincluded in subsequent analyses. Lack of genomic DNA con-tamination was confirmed by PCR amplification of RNAsamples followed by an electrophoresis on a 1% agarosegel. A conventional RT-PCR was made to control the purityof the band of housekeeping gene and target Hd86 gene.
2.3. Identification of housekeeping and target genes andtheir primers
The elongation factor 1-alpha (ELF1A) was used as ahousekeeping gene as described by Nijhof et al. (2009,2010). Hd86 primers were created using PerlPrimer v1.1.18and designed from Hd86-A1 sequence (GenBank accessionnumber HQ872020) isolated by Ben Said et al. (2012). Allinformation on ELF1A and Hd86 genes and these primersare shown in Table 2.
2.4. Determination of Hd86 expression levels
cDNA was be synthesized from 500 ng of DNA-free RNAisolated from rabbit blood and 8 H. scupense life stagesas previously described using the SuperScript First-StrandSynthesis System for RT-PCR (Invitrogen) according to themanufacturer’s directions and stored at −20 ◦C until use.
A quantitative RT-PCR assay using SYBR® green detec-tion was designed and optimized for the transcriptionprofiling of housekeeping ELF1A. Real-time analysis wascarried out on an ABI PRISM 7700 (Applied Biosystems).RT-PCR amplification mixtures (25 �l) were prepared asfollows, cDNA generated from 5 ng of RNA template, 12.5 �lSYBR® green PCR master Mix (2×) (Applied Biosystems),
NA expression profile in Hyalomma scupense life stages,cy between adult and immature instars? Vet. Parasitol.
900 nM forward and reverse primers for ELF1A housekeep-ing gene and 100 nM forward and reverse primers forHd86 target gene. The cycling conditions comprised a 5 mindenaturation and polymerase activation step at 95 ◦C, 40
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Tab
le
2D
etai
ls
of
the
quan
tita
tive
RT-
PCR
of
Hd8
6
targ
et
gen
e
and
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A
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ren
ce
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e
use
d
in
the
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t
stu
dy.
Sym
bol
Gen
e
nam
e
Fun
ctio
n
Forw
ard
pri
mer
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erse
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Am
pli
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len
gth
Effi
cien
cy
Gen
Ban
k
acce
ssio
nn
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Ref
eren
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on
fact
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ha
Com
pon
ent
of
the
euka
ryot
ictr
ansl
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nal
app
arat
us
5′ -C
GC
AA
GTC
TGG
CA
AG
TCTG
A-3
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AC
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2010
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86
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86
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know
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5′ -G
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PRESSitology xxx (2013) xxx– xxx 3
cycles of 95 ◦C for 10 s, 60 ◦C for 30 s and 72 ◦C for 30 s.Upon completion of the amplification program, a dissoci-ation analysis (52–95 ◦C) was performed to determine thepurity of the PCR amplicons.
To estimate amplification efficiencies, a standard curvewas generated for each primer pair based on known quan-tities of cDNA for H. scupense (10-fold serial dilutionscorresponding to cDNA transcribed from 50 ng to 0.05 ngof total RNA in triplicate) and analyzed using the SequenceDetector V.1.6.3 software (Applied Biosystems). All assaysincluded this standard curve, a no-template control andeach of the test cDNAs. Primers, amplicon lengths and PCRefficiencies are indicated in Table 2.
Raw Ct values were transformed to quantities using thecomparative Ct method and specific PCR efficiencies. TheHd86 expression levels were normalized using the geomet-ric mean of selected reference gene quantities in MicrosoftExcel and the 95% confidence interval was calculated.Differential gene expression was considered significantwhen the 95% confidence interval of the mean normalizedexpression levels did not overlap (equivalent to p < 0.05)(Table 3).
3. Results
3.1. Quantitative RT-PCR
The efficiencies of the quantitative RT-PCR of ELF1Aand Hd86 mRNA were uniformly high and estimated at105% and 103%, respectively, making all assays suitablefor quantitative analysis (Table 2). All samples generateda single band and the absence of primer dimer formationwas confirmed by a dissociation assay performed with eachassay (data not shown). None of the primer combinationsamplified cDNA synthesized from rabbit blood RNA, whichexcludes the interference of the rabbit RNA in PCR assay.
Raw Ct values of ELF1A housekeeping gene ranged from16.83 to 19.16 while those for Hd86 target gene ranged from19.82 to 24.32. Consequently, The Ct variation between allsamples was 2.33 for ELF1A and 4.50 for Hd86.
3.2. Hd86 expression analysis
The expression profile of Hd86 mRNA in H. scupense wasobtained by normalizing its expression with ELF1A refer-ence gene (Fig. 1). It should be noted that all samples werecollected at a single well defined points from each life stageand fluctuations possibly occurring during these life stagescould therefore have been missed. Hd86 expression wasdetected at high level in unfed larvae before setting on rab-bit and decreased by 5.01-fold (3.02–8.08) (p = 0.019) afterfeeding (Fig. 1 and Table 3).
After molting of engorged larvae to unfed nymphs,Hd86 expression level increased (∼5-fold; p = 0.049) tolevels similar to that found in unfed larvae. The feedingof nymphs did not generate significant modifications onHd86 mRNA expression level. After molting of engorged
NA expression profile in Hyalomma scupense life stages,cy between adult and immature instars? Vet. Parasitol.
nymphs to unfed adults, expression level decreased sig-nificantly by 12.78 (10.77–17.39) (p < 0.001) and 9.25(5.77–15.72)-fold (p < 0.001), in unfed males and unfedfemales, respectively. When compared to unfed nymphs,
4 M. Ben Said et al. / Veterinary Parasitology xxx (2013) xxx– xxx
LF1A ref
Fig. 1. Hd86 expression levels in several life stages normalized against Einterval of the normalized expression.
Hd86 expression levels were 13.82 (5.39–24.45) (p = 0.035)and 9.93 (2.87–22.08)-fold (p = 0.038) lower in unfed malesand females, respectively (Fig. 1 and Table 3).
Following feeding of adults, Hd86 expression levelincreased, approximately, by 9.53 (6.25–12.29) (p = 0.012)and 2.95 (0.72–5.87)-fold (p = 0.158), in partially fed malesand partially fed females, respectively. The expressionlevel of Hd86 was twice higher in adult males com-pared to females (p = 0.120). Comparatively to the engorgednymphal stage, the expression of Hd86 in partially fedmale and female ticks remains lower by approximately1.34 (0.87–2.16) (p = 0.286) and 3.13 (1.75–7.94)-fold(p = 0.015), respectively (Fig. 1 and Table 3).
4. Discussion
In a previous report, we carried out an experimen-tal vaccination trial with rHd86 against H. scupense ticksinfestations in cattle (Galaï et al., 2012). The most surpris-ing result was the discrepancy between the effect of theHd86 vaccine against larvae and adult H. scupense infesta-tions (Galaï et al., 2012). Several hypotheses were advancedto explain this result, among which a differential Hd86expression level during H. scupense life stages was sug-gested. In order to explore this hypothesis, the normalizedHd86 mRNA expression levels were analyzed in several H.scupense life stages (Fig. 1).
Our results showed that the Hd86 mRNA expres-sion profile fluctuated significantly within H. scupense lifestages. In juvenile stages, a significant reduction of thetranscript number was noted during feeding and particu-larly during molting, while the Hd86 transcript number wasincreased significantly after feeding in adult stages. Thesevariations were more pronounced than those observed inthe Bm86 mRNA expression level in R. microplus and weresimilar to Ra86 mRNA expression level, the homologue ofBm86 in Rhipicephalus appendiculatus (Nijhof et al., 2009).The observed difference between Bm86 and Hd86 expres-sion profiles may be associated to life cycle of the two tickspecies. In fact, H. scupense is a two-host tick that takes one
Please cite this article in press as: Ben Said, M., et al., Hd86 mRcould it contribute to explain anti-tick vaccine effect discrepan(2013), http://dx.doi.org/10.1016/j.vetpar.2013.07.035
year to complete its various life stages under the climaticconditions of North Africa (Walker et al., 2013). In contrast,R. microplus feeds continuously on a unique host withoutany rest period between life stages (Walker et al., 2013).
erence gene expressed in H. scupense. Bars represent the 95% confidence
Moreover, the entire feeding cycle of R. microplus can becompleted in 3 weeks (Walker et al., 2013).
It should be noted that little is known about Bm86 pro-tein function and specific cell type location. The Bm86midgut protein is located predominantly on the microvillisurface of digest cells and its thought that it may play a rolein the cell growth regulation based on its sequence andstructural homology to epidermal growth factor precur-sors (Gough and Kemp, 1993; Nijhof et al., 2010). Indeed,the high Hd86 expression levels found in unfed larvae andunfed nymphs support this hypothesis.
It has been suggested that Bm86 protein could have arole in endocytosis of the blood meal (Kemp et al., 1989),but the low levels of Hd86 expression in fed larvea andpartially fed female and the comparable expression lev-els between unfed and fed nymphs leave this suggestionless probable since the expression of proteins involved inendocytosis is expected to increase during the uptake ofa blood meal (Liyou et al., 1996; Sojka et al., 2013). How-ever, several studies have shown that after absorption ofhemoglobin in the blood meal, R. microplus digestive cellsdetach from the basal lamina in the gut lumen and becomemotile (Walker and Fletcher, 1987; Agyei and Runham,1995). Exhausted digestive cells are thus replaced by otherundifferentiated cells where the rate of the Bm86 pro-tein is very low (Sonenshine, 1991; Nijhof et al., 2009).Consequently, the consecutive cycles of growth and differ-entiation of these cells lead to the presence of digestive cellsat different stages of differentiation. This finding could beone of the main causes of the Hd86 mRNA expression leveldecrease in larvae after feeding.
Besides, the most interesting result was noted aftermolting of engorged nymphs to unfed adults. Indeed,Hd86 expression level decreased significantly after nymphsmolting to adults. Moreover, when unfed and fed femaleadult tick stages were compared, the expression level ofHd86 was not statistically different whereas Hd86 expres-sion levels in both unfed and fed H. scupense nymphsremained both high and stable. These findings might con-tribute to understand why immature stages, and morelikely the nymphs, represent the main Hd86 vaccine tar-
NA expression profile in Hyalomma scupense life stages,cy between adult and immature instars? Vet. Parasitol.
get in H. scupense ticks (Galaï et al., 2012). Similar datawere reported in R. appendiculatus. In fact, high Ra86 mRNAexpression levels were found in unfed and fed immatureticks and decreased significantly in adults (Nijhof et al.,
Please cite this article in press as: Ben Said, M., et al., Hd86 mRcould it contribute to explain anti-tick vaccine effect discrepan(2013), http://dx.doi.org/10.1016/j.vetpar.2013.07.035
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Tab
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3p-
Val
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of
the
Hd8
6
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.
PRESSitology xxx (2013) xxx– xxx 5
2009). Interestingly, Olds et al. (2012) demonstrated thatthe effect of cattle Ra86 vaccination is mainly expressedagainst nymphal stage as the vaccine significantly impededthe nymphal to adult molting. However, further studies areneeded in order to evaluate separately the effect of Hd86vaccinated against nymphs and larvae of H. scupense andto confirm our RT PCR results by quantification of Hd86protein in the three tick instars.
The present study has shown that H. scupense malesexpressed higher levels of Hd86 than females, for this rea-son male ticks it could be hypothesized that they might bemore susceptible to the Hd86 vaccination effect althoughthe lesser blood volume they take could impede vaccineefficacy. In practice, the reproductive capacity of H. scu-pense male ticks has not been altered by Hd86 vaccinationas no significant differences in female ticks egg-layingcapacity whether fed on vaccinated or control calves werereported (Galaï et al., 2012).
5. Conclusion
The expression profile of Hd86 mRNA demonstrated asignificant fluctuation within life stages of H. scupense. Theobserved differences in Hd86 expression profile betweenjuvenile and adult stages may explain, in part, the Hd86vaccine effect discrepancy between these two life stages.In order to confirm these data, Hd86 protein quantificationin H. scupense during these various developmental stagesshould be investigated.
Competing interests
The authors declare that they have no competing inter-ests.
Authors’ contributions
M.B.S. designed and performed molecular biologyexperiments, analyzed the data, and wrote the manuscript.Y.G. participated in design study and draft the manuscript.M.B.A. advised on Quantitative RT-PCR. J.F. helped to draftthe manuscript. M.J. assisted to obtain the different tickstages. M.A.D. conceived the study, and participated in itsdesign and coordination and drafted the manuscript. Allauthors read and approved the final manuscript.
Acknowledgments
This study was supported by a Wellcome Trust AnimalHealth Initiative in the Developing World grant entitled“Adapting Recombinant Anti-Tick Vaccines to Livestockin Africa” (Wellcome Trust Project number: 075799) andby the “Laboratoire d’Epidémiologie des Maladies Enzoo-tiques des Herbivores en Tunisie” (LR02AGRO3) funded bythe Ministry of High Education and Scientific Research of
NA expression profile in Hyalomma scupense life stages,cy between adult and immature instars? Vet. Parasitol.
Tunisia. The authors are grateful to Limam Sassi, TaoufikLahmar and Béchir Gesmi, from the National School ofVeterinary Medicine of Sidi Thabet (Tunisia), for their tech-nical assistance.
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