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Vaccine 29 (2011) 2248–2254

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

ontrol of Rhipicephalus (Boophilus) microplus infestations by the combination ofubolesin vaccination and tick autocidal control after subolesin gene knockdownn ticks fed on cattle

ctavio Merinoa, Consuelo Almazána, Mario Canalesb, Margarita Villarb, Juan A. Moreno-Cidb,gustín Estrada-Penac, Katherine M. Kocand, José de la Fuenteb,d,∗

Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Tamaulipas, Km. 5 carretera Victoria-Mante, CP 87000 Ciudad Victoria, Tamaulipas, MexicoInstituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, SpainDepartment of Parasitology, Veterinary Faculty, Miguel Servet 177, 50013 Zaragoza, SpainDepartment of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA

r t i c l e i n f o

rticle history:eceived 22 November 2010eceived in revised form 4 January 2011ccepted 18 January 2011vailable online 1 February 2011

a b s t r a c t

Tick subolesin was shown in immunization trials using the recombinant protein to protect hosts againsttick infestations. In this study, we demonstrated that subolesin vaccination and release of ticks after sub-olesin knockdown by RNA interference (RNAi) could be used for the control of Rhipicephalus (Boophilus)microplus tick infestations in cattle and suggested that the combination of these methods could increase

eywords:attle tickoophilusaccineNA interference

the efficacy of cattle tick control under some circumstances. The greatest tick control was obtained whenboth release of ticks after subolesin knockdown and vaccination were used concurrently. However, mod-eling results suggested that vaccine efficacy could be increased if at least 80% of the ticks infesting cattlecorrespond to subolesin-knockdown ticks. The results of this proof-of-concept trial demonstrated the effi-cacy of the sterile acarine technique (SAT) through production of subolesin-knockdown larvae by dsRNAinjection into replete females for the control of R. microplus tick infestations, alone or in combination

n.

ubolesin with subolesin vaccinatio

. Introduction

Rhipicephalus (Boophilus) microplus ticks are distributed in trop-cal and subtropical regions of the world with geographic rangexpansion due to changes in climatic conditions and host pop-lations increase and movement [1–5]. Infestations with theattle tick, R. microplus, economically impact cattle production byeducing weight gain and milk production, and by transmittingathogens that cause babesiosis (Babesia bovis and Babesia bigem-

na) and anaplasmosis (Anaplasma marginale) [6].The use of acaricides constitutes a major component of inte-

rated tick control strategies [7]. However, acaricide applicationas had limited efficacy in reducing tick infestations and is often

ccompanied by serious drawbacks, including the selection ofcaricide-resistant ticks, environmental contamination and con-amination of milk and meat products with drug residues [7]. All ofhese issues reinforce the need for alternative approaches to control

∗ Corresponding author at: Instituto de Investigación en Recursos CinegéticosREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, Spain.

E-mail addresses: jose delafuente@yahoo.com, djose@cvm.okstate.eduJ. de la Fuente).

264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2011.01.050

© 2011 Elsevier Ltd. All rights reserved.

cattle tick infestations, which may include the use of hosts naturallyresistant to ticks, pheromone-impregnated decoys for attractingand killing ticks, biological control methods and vaccines [8–10].

Vaccines were developed and commercialized in the early 1990sthat induced immunological protection in cattle against cattle tickinfestations. These commercial vaccines, Gavac and TickGARD, con-tained the recombinant R. microplus BM86 gut antigen [11]. Thesevaccines reduce the number of engorging female ticks, their weightand reproductive capacity. Thus the greatest vaccine effect wasthe reduction of larval infestations in subsequent generations. Vac-cine controlled field trials in combination with acaricide treatmentsdemonstrated that an integrated approach resulted in controlof cattle tick infestations while reducing the use of acaricides[11–13]. These trials demonstrated the advantages of tick controlby vaccination, by being cost-effective, reducing environmentalcontamination and preventing the selection of drug resistant ticksthat result from repeated acaricide application. In addition, thesevaccines also reduced or prevented the transmission of pathogens

by reducing tick populations and/or affecting tick vector capacity[11,12,14].

Subolesin was discovered in Ixodes scapularis as a tick protectiveantigen [15] and then characterized in several tick species includ-ing R. microplus [16–19]. Immunization trials with recombinant

cine 29

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ubolesin resulted in protection of hosts against tick infestationsy decreasing tick survival and reproduction rates [18–20,23]. Inddition, subolesin knockdown by RNAi had a profound effect onick biology and caused degeneration of tick tissues (guts, salivarylands, reproductive tissues and embryos). In subsequent stud-es, subolesin was found to control tick gene expression, impacthe innate immune response, and decrease the vector capacityf ticks for A. marginale and A. phagocytophilum [16–19,21–27].otably, subolesin knockdown by RNAi resulted in productionf sterile ticks. When sterile female or male ticks were pairedith normal ticks, lack of successful mating by the sterile ticksrevented female engorgement and oviposition. Consequently,he release of subolesin-silenced ticks was proposed as a sterilecarine technique (SAT) for autocidal control of tick populations28].

The combination of different control methods adapted to geo-raphic areas may be the most efficient way to control ticknfestations and pathogen transmission [9,10]. The combinationf tick vaccines with other control methods may be required toffect maximum cattle tick control. The experiments reported hereere conducted to characterize the effect on cattle tick infestations

f the combination of subolesin vaccination with tick autocidalontrol through subolesin gene knockdown by RNAi. The resultsemonstrated that the combination of cattle tick vaccines and SAThow promise as an effective and safe approach for cattle tickontrol.

. Materials and methods

.1. Ticks

The R. microplus (Susceptible Media Joya strain, CENAPA,exico) ticks were obtained from a laboratory colony maintained

t the University of Tamaulipas, Mexico. Originally, these ticks wereollected from infested cattle in Tapalpa, Jalisco, Mexico. Tick lar-ae were fed on cattle and collected after repletion to allow forviposition and hatching in humidity chambers at 12 h light:12 hark photoperiod, 22–25 ◦C and 95% relative humidity (RH). Larvaeere used for infestations at 15 days after hatching from eggs.

.2. RNA interference in ticks

Oligonucleotide primers containing T7 promoterequences (in italics) at the 5′-end (SUB: D8BMT75: 5′-AATACGACTCACTATAGGGTACTGACTGGGACCCCTTGCACAGT-3′

nd D8BMT73: 5′-TAATACGACTCACTATAGGGTACTCGAGTTTGGTAG-TAGCACA-3′) were synthesized and used for in vitro transcriptionf subolesin (Genbank accession number DQ159966) dsRNA usinghe Acess RT-PCR system (Promega, Madison, WI, USA) and the

egascript RNAi kit (Ambion, Austin, TX, USA) following man-facturer’s recommendations [18]. The dsRNA was purified anduantified by spectrophotometry.

Engorged R. microplus females were weighted and injected with�l of dsRNA (5 × 1010 molecules/�l) in the right spiracular plateithin 6 h after dropping off the host. Ticks were injected usingHamilton syringe with a 1-in., 33 gauge needle. Control ticksere injected with the unrelated GIII dsRNA. The GIII sequence was

dentified in R. microplus and was shown previously to not affectick feeding, mortality and oviposition after RNAi [18]. After dsRNAnjection, ticks were stored individually in an incubator at 22–25 ◦Cnd 95% RH for oviposition. The subsequently hatched larvae were

se for cattle infestation.

Subolesin expression silencing was characterized in adultemale ticks obtained from larvae produced by subolesin dsRNA-njected replete females and GIII dsRNA-injected controls. Ten tickser group were dissected and whole internal tissues pooled for

(2011) 2248–2254 2249

RNA extraction to evaluate gene expression by semi-quantitativeRT-PCR using the same oligonucleotide primers used for subolesindsRNA synthesis. The mRNA levels (nanograms per amplicon) wereestimated in ethidium bromide-stained agarose gels by comparisonto a standard curve of the target gene, normalized against tick actinmRNA levels [17] and compared between subolesin gene dsRNAand GIII dsRNA-injected control ticks to calculate the percent ofgene silencing after RNAi.

2.3. Cattle vaccination with recombinant subolesin

Recombinant R. microplus subolesin was expressed inEscherichia coli from a synthetic gene (Genbank accession numberGQ456170) optimized for codon usage in E. coli and purified by Niaffinity chromatography to 80–90% purity. Protein adjuvation wasdone by mixing a solution of anhydromannitoletheroctodecenoate(Montanide ISA 50 V; Seppic, Paris, France) with the recombinantprotein solution in batch-by-batch processes using a high-speedmixer Heidolph DIAX 900 (Heidolph Elektro, Kelheim, Germany)at 8000 rpm and the vaccine was filled manually under sterileconditions in glass bottles of 20 ml (Wheaton, Millville, NJ, USA) ata concentration of 100 �g/2 ml dose. Quality controls were madeby testing mechanical and thermal stability of vaccine emulsionsas described previously [29].

Seven-month-old European crossbred calves were immunizedwith 3 doses (days 0, 28 and 49) containing 100 �g/dose of purifiedrecombinant subolesin formulated as described above. Negativecontrols were injected with adjuvant/saline alone. Cattle wereinjected intramuscularly with 2 ml/dose using a 5 ml syringe andan 18G needle.

2.4. Experimental design, data collection and analysis

Cattle were randomly assigned to two experimental groups of6 animals each, subolesin-vaccinated and adjuvant/saline control.Thirty days after the last immunization (day 79), cattle in both vac-cinated and control groups were infested with R. microplus larvae.Three tick infestations treatments were evaluated on each animal inindividual cells glued on the back of the calf. The cells were infestedwith 500 larvae obtained from a replete female injected with GIIIcontrol dsRNA (control), 500 larvae obtained from a replete femaleinjected with subolesin dsRNA (RNAi) and a combination of 500control and 500 RNAi larvae (mixed). Cattle were cared for in accor-dance with standards specified in the Guide for Care and Use ofLaboratory Animals for the University of Tamaulipas.

Adult engorged female ticks dropping from cattle were dailycollected, counted and weighted. All the collected adult femaleticks were assessed for oviposition [30]. The personnel collectingthe ticks were ‘blinded’ as to which group animals belonged. Theeffect of each treatment on cattle tick infestations was evaluatedemploying the formulae used before in tick vaccine experiments[18,20,23,31].

Effect on the number of adult female ticks (DT) = 100[l − (NTV/NTC)], where NTV is the number of adult female ticks inthe vaccinated group and NTC is the number of adult female ticksin the control group.

Effect on tick weight (DW) = 100 [1 − (WTV/WTC)], where WTVis the average adult female tick weight in the vaccinated group andWTC is the average adult female tick weight in the control group.

Effect on oviposition (DO) = 100 [1 − (PATV/PATC)], where PATVis the average weight of the eggs per survived tick in the vaccinated

group and PATC is the average weight of the eggs per survived tickin the control group.

For each evaluated parameter, the percent reduction was cal-culated with respect to the adjuvant/saline injected control cattle(DT1, DW1, DO1) and the tick control group (DT2, DW2, DO2)

2250 O. Merino et al. / Vaccine 2

Fig. 1. Experimental design and data analysis. Cattle were randomly assigned to twoexperimental groups (N = 6 per group), subolesin-vaccinated and adjuvant/salinecontrol and infested with R. microplus larvae. Three tick infestation treatments wereevaluated on each animal in individual cells containing 500 control larvae (con-trol), 500 larvae obtained from a replete female after subolesin knockdown by RNAi(RNAi) and a combination of 500 control and 500 RNAi larvae (mixed). The percentreduction was calculated for tick numbers (DT), tick weight (DW) and oviposition(DO) with respect to the adjuvant/saline control cattle (DT1, DW1, DO1) and the tickcvR

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ontrol group (DT2, DW2, DO2) and were statistically compared between subolesin-accinated and control groups (1c, 1r and 1m) and within each treatment betweenNAi and mixed treatments and control ticks (2r, 2rc, 2m, and 2mc).

Fig. 1). The average ± S.D. for adult female tick number, weightmg), and oviposition (egg weight (mg)/tick) were calculated andompared between subolesin-vaccinated and control cattle andetween RNAi and mixed treatments and control ticks by �-test˛ = 0.01) for tick numbers and by Student’s t-test with unequalariance (P = 0.05) for tick weight and oviposition.

Treatment efficacy was calculated as 100 [l − (CRT × CR0)],here CRT = NTV/NTC and CR0 = PATV/PATC represent the reduc-

ion in the number of adult female ticks and oviposition asompared to the control ticks fed on adjuvant/saline injected con-rol cattle, respectively.

.5. Determination of serum antibody levels by ELISA

Before each immunization and tick infestation and at the end ofhe experiment (day 95), blood samples were collected from eachalf into sterile tubes and maintained at 4 ◦C until arrival at the lab-ratory. Serum was then separated after centrifugation and storedt −20 ◦C. Serum antibody titers were determined using an antigen-pecific indirect ELISA. Purified subolesin antigen (0.1 �g/well)as used to coat ELISA plates overnight at 4 ◦C. Sera were seri-

lly diluted to 1:10, 1:100 and 1:1000 in PBST (PBS/0.5% Tween0, pH 7.2) and 10% fetal bovine serum (Sigma). The plates were

ncubated with the diluted sera for 1 h at 37 ◦C and then incu-ated with 1:10,000 rabbit anti-bovine IgG-HRP conjugates (Sigma)or 1 h at 37 ◦C. The color reaction was developed with 3,3′,5,5′-etramethylbenzidine (Sigma) and the OD450nm was determined.fter incubation the plates were washed with PBST. Antibody titersere considered positive when they yielded an OD450nm value at

east twice as high as the preimmune serum. Antibody titers inmmunized cattle were expressed as the OD450nm value for theighest serum dilution (1:1000) and compared between vaccinated

nd control cattle using an ANOVA test (P = 0.05). A correlation anal-sis was conducted in Microsoft Excel (version 12.0) to comparehe numbers of female ticks collected after feeding with anti-ubolesin antibody titers at time of tick infestation in individualattle.

9 (2011) 2248–2254

2.6. Model simulations of subolesin vaccination and RNAi oncattle tick control

The simulations were done using Simgua (www.simgua.com).This is a no-nonsense modeling application for dynamics simula-tions. The objective was to simulate the variations in the abundanceof ticks as a consequence of the combined effect of the subolesinvaccination and infestation with ticks after subolesin knockdownby RNAi to control the wild type cattle tick population. In the model,the host population had a fixed growth rate of 1.5 with growth lim-its imposed by a variable carrying capacity of the environment (k).The variability of k was simulated with a sinusoidal curve to simu-late temporal stochasticity. The tick population was modeled withthe same growth pattern, but the carrying capacity (kt) was lim-ited by the number of available hosts with an upper limit of 1000ticks/host. The death rate of both hosts and ticks was regulatedby the k and kt limits along the time. Other effects of climate onticks and tick mortality were considered as described by Corsonet al. [32]. Mortality derived from density-dependent effects [33]was not included since the model was intended only to extend theresults obtained from the proof-of-concept experiment describedhere using subolesin vaccination and release of ticks after subolesinknockdown by RNAi.

3. Results and discussion

The development of new methods to control cattle tickinfestations and reduce the incidence of bovine anaplasmosisand babesiosis while minimizing acaricide applications is essen-tial towards improving cattle health and production in tropicaland subtropical regions of the world. Tick vaccines based onR. microplus BM86/BM95 antigens have proven their efficacyfor control of cattle tick infestations and the transmission oftick-borne pathogens in some regions [11]. Field application ofcattle tick vaccines demonstrated that the combination of dif-ferent control methods adapted to a geographic area may bethe most efficient way to control tick infestations and pathogentransmission [9,10]. Based on these premises, the experimentsreported herein were conducted to characterize the impact of thecombination of subolesin vaccination and tick autocidal controlafter subolesin gene knockdown by RNAi on cattle tick infesta-tions.

3.1. Effect of subolesin vaccination on the control of cattle tickinfestations

Anti-subolesin antibody titers raised in vaccinated cattle afterthe first immunization and remained significantly higher through-out the experiment when compared to adjuvant/saline injectedcontrols (P < 0.01; Fig. 2).

The effect of subolesin vaccination on the control of R. microplustick infestations was characterized by comparing the results withcontrol ticks between subolesin-vaccinated and adjuvant/salinecontrol cattle (DT1c, DW1c, DO1c; Table 1). As demonstrated pre-viously [18], the results showed a significant reduction in ticknumbers but no effect on tick weight and oviposition (Table 1).The overall subolesin vaccine efficacy (E1) was 44% considering theeffect on tick numbers and oviposition (Fig. 3).

When antibody titers at tick infestation time (day 79) were cor-

related with the number of control ticks collected after feeding,a positive correlation was obtained (R2 = 0.7; Fig. 4). These resultsstrongly suggested, as in previous experiments with BM86 [12,34],that the reduction in cattle tick infestations were the result of anti-subolesin antibodies in vaccinated cattle.

O. Merino et al. / Vaccine 29 (2011) 2248–2254 2251

Table 1Effect of different treatments on the control of R. microplus infestations in cattle.

Vaccinea Treatmentb R. microplus (susceptible; Mexico strain)

Percent reduction (average ± S.D.)c

DT DW DO

Subolesin Control DT1c = 42%* DW1c = 2% DO1c = 3%– – –(18 ± 6) (286 ± 18) (126 ± 14)

RNAi DT1r = 13%*DT2r = 40%*

DW1r = 0% DO1r = 0%

(11 ± 4) DW2r = 26%* DO2r = 25%**(212 ± 31) (94 ± 46)

Mixed DT1m = 24%* DW1m = 5% DO1m = 14%*DT2m = 0% DW2m = 13%* DO2m = 18%*(31 ± 6) (249 ± 26) (103 ± 10)

Adjuvant/saline control Control – – –(32 ± 6) (293 ± 19) (129 ± 7)

RNAi – – –DT2rc = 60%* DW2rc = 33%* DO2rc = 44%*(13 ± 4) (195 ± 21) (72 ± 17)

Mixed – – –DT2mc = 8% DW2mc = 10%* DO2mc = 8%**(41 ± 12) (263 ± 16) (119 ± 11)

a Cattles were randomly assigned to two experimental groups (N = 6 per group), subolesin-vaccinated and adjuvant/saline control and infested with R. microplus larvae.b Three tick infestation treatments were evaluated on each animal in individual cells containing 500 control larvae (control), 500 larvae obtained from a replete female

after subolesin knockdown by RNAi (RNAi) and a combination of 500 control and 500 RNAi larvae (mixed).l catt

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down after dsRNA injection into replete females continues untillarval stages but suggested an RNAi dilution effect that could pre-vent gene knockdown in subsequently molted nymphs and adults[17,24,35]. Herein, the results showed an effect of subolesin gene

c The percent reduction was calculated with respect to the adjuvant/saline controick infestation; DW, % reduction in tick weight; DO, % reduction in oviposition. Innd oviposition (egg weight (mg)/tick) and were compared between subolesin-vacc-test (*˛ < 0.005) for tick number and by Student’s t-test with unequal variance (*P

.2. Effect of subolesin gene knockdown on cattle tick infestations

The effect of subolesin knockdown by RNAi on R. microplus ticknfestations was characterized by comparing the results betweenarvae obtained from replete females injected with subolesinsRNA and control ticks fed on vaccinated (DT2r, DW2r, DO2r;able 1) and adjuvant/saline control (DT2rc, DW2rc, DO2rc; Table 1)attle. A significant decrease in tick numbers, weight and ovipo-ition was observed, and the results were similar for ticks fed

n subolesin-vaccinated and adjuvant/saline control cattle (com-are DT2r, DW2r, DO2r with DT2rc, DW2rc, DO2rc, respectively;able 1). These results were similar to those reported previouslyor subolesin knockdown in R. microplus [17,18]. However, this was

ig. 2. Antibody response in vaccinated and adjuvant/saline injected cattle. Bovineerum antibody titers to recombinant subolesin were determined by ELISA in cattleaccinated with subolesin and adjuvant/saline controls. Antibody titers in immu-ized cattle were expressed as the OD450nm value for the 1:100 serum dilution andompared between vaccinated and control cattle using an ANOVA test (*P < 0.01).he time of vaccination shots and tick infestation (arrows) are indicated.

le (DT1, DW1, DO1) and the tick control group (DT2, DW2, DO2): DT, % reduction inhesis are shown the average ± S.D. for adult female tick number, tick weight (mg),

d and control groups and between RNAi and mixed treatments and control ticks by1; **P < 0.05) for tick weight and oviposition.

the first experiment characterizing the effect of subolesin dsRNAinjection into replete females on the subsequent generation ofadult ticks. Previous experiments have shown that gene knock-

Fig. 3. Efficacy of each treatment on the control of cattle tick infestations consid-ering the effect on tick numbers and oviposition. Treatment efficacy was calculatedas 100 [l − (CRT × CR0)], where CRT = NTV/NTC and CR0 = PATV/PATC represent thereduction in the number of adult female ticks and oviposition as compared to thecontrol ticks fed on adjuvant/saline injected control cattle, respectively. Treatmentefficacy was calculated for subolesin vaccination (E1), subolesin vaccination andrelease of 100% larvae after subolesin gene knockdown (E2), and subolesin vacci-nation and release of 50% larvae after subolesin gene knockdown mixed with 50%control ticks (E3).

2252 O. Merino et al. / Vaccine 2

Fig. 4. Anti-subolesin antibody titers positively correlated with the reduction of ticki1st

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nfestations. A correlation analysis was conducted using Microsoft Excel (version2.0) between the number of control female ticks collected after feeding from bothubolesin-vaccinated and control cattle and anti-subolesin antibody titers at time ofick infestation in individual cattle. The linear correlation coefficient (R2) is shown.

nockdown on tick female weight and oviposition (Table 1), thusuggesting that at least in the one-host tick, R. microplus, genenockdown continued until adult tick stages after dsRNA injectionnto replete females. In fact, when subolesin mRNA levels wereompared between adult female ticks that developed from lar-ae produced by subolesin dsRNA-injected replete females and GIIIsRNA-injected controls, gene expression silencing averaged (±SD)8 ± 33% (range, 0–90%) and was similar for ticks fed on vaccinated48 ± 38; range, 0–80%) and control (48 ± 32; range, 0–90%) cattle.

.3. Effect of subolesin vaccination and gene knockdown on theontrol of cattle tick infestations

The main objective of this experiment was to characterize theffect of combining subolesin vaccination and release of ticks afterubolesin knockdown by RNAi on the control of R. microplus ticknfestations in cattle. Although a significant added effect of sub-lesin vaccination was observed only in the reduction of tickumbers for subolesin-knockdown ticks fed on vaccinated cattlehen compared to ticks fed on adjuvant/saline controls (DT1r;

able 1), the efficacy of the combined treatment (E2) was 75% (66%nd 27% reduction in the number of ticks and oviposition whenompared to the control ticks fed on adjuvant/saline control cattle,espectively) (Fig. 3).

The efficacy of the combined treatment with subolesin vac-ination and the release of ticks after subolesin knockdown byNAi was 1.7-fold higher than the efficacy of the subolesin vaccinelone. However, under natural conditions, the ticks with sub-lesin gene knockdown would mix with wild type ticks infestinghe same animal. To mimic this situation, subolesin-knockdownarvae were mixed 1:1 with control larvae and fed on vacci-ated and control cattle. The results showed a reduction in tick

emale weight and oviposition when compared to control ticksed on both vaccinated and control cattle (DW2m, DO2m, andW2mc, DO2mc; Table 1). However, this effect was less pro-ounced than when subolesin-knockdown larvae were fed alonen both vaccinated and control cattle (compare DT2r, DW2r, DO2r,T2rc, DW2rc, DO2rc with DT2m, DW2m, DO2m, DT2mc, DW2mc,O2mc, respectively; Table 1). Although subolesin RNAi affects

oth female and male fertility [28], the results of female tick weightnd oviposition suggested that some of the control males mateduccessfully with control females and thus produced ticks with nor-al weight and oviposition. Further support for this suggestion was

rovided by the fact that the number of female ticks obtained from

9 (2011) 2248–2254

mixing subolesin-knockdown larvae with control larvae and fed onadjuvant/saline control cattle with a weight higher than the aver-age weight of control ticks (293 mg; Table 1) was 93. Thus, at least38% (93/245) of the female ticks in this group could be assumedto reflect the mating of control female by control male ticks. Nev-ertheless, as expected from previous experiments, the weight ofmost female ticks probably reflected mating between treated andtreated/control ticks [28].

Remarkably, a significant effect on the reduction of tick num-bers and oviposition was observed for subolesin-knockdown ticksmixed with control ticks fed on subolesin-vaccinated cattle whencompared to the same group fed on adjuvant/saline control cat-tle (DT1m and DO1m; Table 1). This effect resulted in an efficacyof the combined treatment for subolesin-knockdown ticks mixedwith control ticks (E3) of 22% (3% and 20% reduction in the numberof ticks and oviposition when compared to the control ticks fed onadjuvant/saline control cattle, respectively) (Fig. 3).

3.4. Possible use of the combination of subolesin vaccination andSAT for the control of cattle tick infestations

The results reported here showed the possibility of combiningsubolesin vaccination and SAT through release of subolesin-knockdown larvae obtained after RNAi as a method to control cattletick infestations. The limitations of this method are associated,among others discussed for sterile-insect technique (SIT) [36], withthe massive production of ticks with subolesin knockdown and therelease of genetically modified ticks into the environment. How-ever, the injection of dsRNA into replete R. microplus females shownhere and in previous publications [17,18,37] could allow the pro-duction of large amounts of larvae with subolesin gene knockdown.Furthermore, the effect of gene knockdown after RNAi in ticks hasbeen shown to be transient and mechanisms have not been discov-ered that could amplify and maintain this effect through multiplegenerations [35].

The efficacy of the subolesin vaccine shown herein and in pre-vious experiments (44–51%) [18] was lower than that obtainedwith BM86 (60–70%) on the control of the same R. microplus strain[18,31,38]. The efficacy of the combined subolesin vaccination andrelease of ticks after subolesin knockdown by RNAi (75%) was sim-ilar or slightly higher than that obtained with the BM86 vaccine.However, when mixed infestations of subolesin-knockdown andcontrol ticks were characterized on subolesin-vaccinated cattle,the efficacy of the treatment decreased to 22%. It has been gen-erally accepted from experimental data and models that a tickvaccine efficacy higher than 50% is sufficient to reduce larval infes-tations in subsequent generations [13,29,39–41]. Therefore, furtherexperiments are needed to characterize the efficacy of the com-bined subolesin vaccination and SAT by increasing the proportionof subolesin-knockdown larvae over wild type ticks to define thenumber of RNAi treated ticks to be released according to the infes-tation load in a particular region.

To address this question, the effect of releasing different num-bers of subolesin-knockdown ticks into the environment wassimulated using the data reported here on tick mortality andoviposition after different treatments in subolesin vaccinated cat-tle (Fig. 5). The simulation was done with subolesin-knockdownticks representing from 10% to 100% of the total tick population inintervals of 10%. To consider stochastic effects and to increase therobustness of the system, 1000 tick generations were simulated

for each combination of subolesin-knockdown and wild type ticks,allowing the release of subolesin-knockdown larvae at each tickgeneration. The efficacy of each treatment on cattle tick control wassimulated in comparison with control ticks fed on adjuvant/salineinjected control cattle.

O. Merino et al. / Vaccine 29 (2011) 2248–2254 2253

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ig. 5. Simulation of tick population control. The figure displays the percent of tontrol cattle. The simulation was conducted with subolesin vaccine only (0% of subnockdown ticks released into the environment. The percent reduction of tick popuas the average of 100 runs.

As expected from results of previous vaccination experimentsnd simulations [11–13,34,39–41], the accumulated efficacy ofach treatment on cattle tick population performance over 1000ick generations (Fig. 5) was higher than treatment efficacy afterne generation only (Fig. 3). In agreement with the experimentalesults obtained here, the highest and lowest treatment efficaciesere obtained when 100% and 50% of subolesin-knockdown ticks

ed on vaccinated cattle, respectively, thus validating the results ofhe simulation. Interestingly, subolesin vaccine efficacy decreaseds the number of subolesin-knockdown ticks released increasedrom 0% to 50% of the tick population (Fig. 5). This negative effectf subolesin-knockdown tick release over tick control could be dueo the fact that the effect of the vaccine decreased in subolesin-nockdown ticks because subolesin levels, targeted by vaccinentibodies, decreased in these ticks and the effect of subolesinnockdown on tick fertility was not sufficient to counteract thisffect until the number of subolesin-knockdown ticks increasedo over 70% of the tick population. In fact, the results of the sim-lation suggested that the efficacy of subolesin vaccination on theontrol of cattle tick infestations could be increased if at least 80% ofhe ticks infesting cattle correspond to subolesin-knockdown ticksFig. 5). This fact may constitute an additional limitation of the com-ination of SAT with subolesin vaccination for cattle tick control,s it requires the release of large numbers of subolesin-knockdownicks into the environment. However, it may still be an option inegions where acaricide-resistant tick populations are present atelatively low infestation rates [11].

. Conclusions

These results demonstrated that the combination of subolesinaccination and release of ticks after subolesin knockdown byNAi could be used for the control of R. microplus tick infestations

n cattle and suggested that the combination of these methodsould increase the efficacy of cattle tick control, at least underome circumstances. The results reported here suggested the usef the SAT through production of subolesin-knockdown larvae bysRNA injection into replete females for the control of R. microplusick infestations, alone or in combination with subolesin vaccina-

ion. However, these experiments are a proof-of-concept only anduture studies are needed to fully address efficacy and safety issuesssociated with the combination of vaccination and SAT using sub-lesin. Additionally, it may be possible to increase the efficacy ofhe subolesin vaccine using different adjuvant/formulations and to

[

[

ntrol with respect to the number of control ticks fed on adjuvant/saline injected-knockdown ticks released) and after a series of growing percentage of subolesin-s shown in the graph was simulated for 1000 tick generations and each simulation

combine the SAT approach with vaccination with BM86 and othertick antigens.

Acknowledgements

We thank Rodolfo Lagunes, Alejandro González and UrielValdez (Universidad Autónoma de Tamaulipas, Mexico) for tech-nical assistance. This work was supported by FOMIX-Tamaulipas,Mexico (project 73622); the Instituto Nacional de Investigación yTecnología Agraria y Alimentaria (INIA), Spain (project FAU2008-00014-00-00) and the Consejería de Educación y Ciencia, JCCM,Spain (project PEII09-0118-8907). M. Villar was funded by the JAE-DOC program (CSIC-FSE), Spain. J.A. Moreno-Cid is a recipient of aJCCM fellowship, Spain.

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