Efficacy of Rhipicephalus (Boophilus) Microplus Bm86 Against Hyalomma

Vaccine 30 (2012) 3453–3458

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Efﬁcacy of Rhipicephalus (Boophilus) microplus Bm86 against Hyalomma dromedarii and Amblyomma cajennense tick infestations in camels and cattle

Manuel Rodríguez-Valle a,∗,1, Amar Taouﬁk b, Mario Valdés c, Carlos Montero a, Ibrahim Hassan d, Shawgi Mohammed Hassan e, Frans Jongejan b,f, Jose de la Fuente g,h a Centro de Ingenieria Genetic and Biotecnologia, Habana, Cuba b Utrecht Centre for Tick-borne Diseases (UCTD), Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3583 CL Utrecht, The Netherlands c Centro Nacional de Parasitología, San Antonio de los Ba˜nos, Habana, Cuba d Department of Microbiology and Parasitology, University of Nyala, Sudan e Department of Parasitology, University of Khartoum, Sudan f Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, PO Box. X04, Onderstepoort 0110, South Africa g Department of Veterinary Pathobiology, Centre for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078-2007, USA h Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, Spain

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Article history: The recombinant Bm86-based tick vaccines have shown their efﬁcacy for the control of cattle ticks, Received 5 January 2012 Rhipicephalus (Boophilus) microplus and R. annulatus infestations. However, cattle ticks often co-exist Received in revised form 13 February 2012 with multi-host ticks such as Hyalomma and Amblyomma species, thus requiring the control of multiple Accepted 8 March 2012 tick infestations for cattle and other hosts. Vaccination trials using a R. microplus recombinant Bm86- Available online 22 March 2012 based vaccine were conducted in cattle and camels against Hyalomma dromedarii and in cattle against Amblyomma cajennense immature and adult ticks. The results showed an 89% reduction in the number of Keywords: H. dromedarii nymphs engorging on vaccinated cattle, and a further 32% reduction in the weight of the Rhipicephalus microplus Hyalomma dromedarii surviving adult ticks. In vaccinated camels, a reduction of 27% and 31% of tick engorgement and egg mass Amblyomma cajennense weight, respectively was shown, while egg hatching was reduced by 39%. However, cattle vaccination Tick with Bm86 did not have an effect on A. cajennense tick infestations. These results showed that Bm86 Vaccine vaccines are effective against R. microplus and other tick species but improved vaccines containing new Bm86 antigens are required to control multiple tick infestations. Cattle © 2012 Elsevier Ltd. All rights reserved. Camels

1. Introduction ferent cattle tick strains [9] and in other tick species [10–14] as Bm86 orthologs. During 1993–1997, two vaccines using recom- Bm86 is a membrane-bound glycoprotein on the surface of R. binant Bm86 were registered for use in Latin American countries microplus gut cells [1]. In 1995, the antigen Bm86 was re-cloned (GavacTM) and Australia (TickGARDTM) [6,15–17] and had similar from an Argentinean strain of R. microplus and designated as Bm95 efﬁcacy for the control of cattle tick infestations [18]. These tick [2]. Antibodies produced in vaccinated cattle bind to Bm86 in the vaccines constitute the only example of commercial vaccines for tick gut and are thought to cause damage mainly by the com- the control of ectoparasites and led the way for research on the plement pathway, but this mechanism is still poorly understood development of vaccines for arthropod vectors of pathogens that [3,4]. The result of Bm86 vaccination is the reduction in the num- affect human and animal health [18]. ber and weight of engorged ticks, the egg laying capacity and the In many regions, R. microplus shares its habitat with other Rhipi- egg fertility, which ultimately reduces tick populations and the cephalus tick species such as R. annulatus, R. decoloratus and R. geigyi prevalence of tick-borne pathogens [5–8]. Bm86 is present in dif- and other multi-host tick species such as those belonging to the Hyalomma and Amblyomma genera which are major constraints to animal production in some regions [19–21]. The camel tick, H. dromedarii is a two host tick distributed in ∗ Corresponding author at: Queensland Alliance for Agriculture & Food Innovation the North, North East, East and Sahara-Sahel regions of Africa that (QAAFI), Queensland Biosciences Precinct, The University of Queensland, DEEDI, GPO occasionally uses three hosts [19,22]. Camels are the principal host Box 6097, St Lucia, Qld 4067, Australia. Tel.: +61 0 7 3255 4529. of the H. dromedarii adults with some records also showing that E-mail address: [email protected] (M. Rodríguez-Valle). cattle and goats are also susceptible [23]. The immature stages can 1 306 Carmody Rd (Building 80 Loading Dock, off Services Road), St. Lucia, Qld 4072, Australia. parasitize rodents, leporids, hedgehogs and birds [23]. In addition,

H. dromedarii is a vector of bacteria [24], protozoa [25], rickettsia of vaccinated and control cattle was determined by ELISA [36]. The [26], and viruses [27]. In the Americas, Rocky Mountain spotted titres of anti-Bm86 IgG were very high in the vaccinated group after fever caused by Rickettsia rickettsii is transmitted by A. cajennese, the last immunization with Bm86, as it is reported in the literature Dermacentor andersoni, D. variablis and R. sanguineus [28]. The Rick- [17]. ettsia amblyommii is transmitted by A. cajennese in Brazil [29]. Ten weeks after vaccination, a cage containing approximately Recombinant Bm86 continues to be the most effective anti- 1500 H. dromedarii larvae (three months old) was glued onto the gen against tick infestations. The protective efficacy of vaccination back of each calf. Engorged nymphs were collected separately from with Bm86-based vaccines has been shown an effect on the con- each calf and reapplied to the same calves in a similar way. Moulted trol of tick infestations by Hyalomma anatolicum anatolicum, R. unfed adult ticks applied were five months old. Tick infestations annulatus, R. decoloratus and H. dromedarii [10,30–32]. Close to were monitored daily. Engorged adults tick were collected, counted 100% control of R. annulatus infestations has been observed using and weighed. Female ticks were incubated at 28 ◦C and 85% relative Bm86 cattle vaccination in different trials [10,30,31,33]. In contrast, humidity for egg laying and weight. Bm86 has shown partial or no protection against some R. microplus strains from different geographic regions and other tick species 2.3. Experiment 2: Bm86 vaccination against H. dromedarii [10,18,30,34,35]. The effect of Bm86 vaccination against different infestations in camels tick species can be further characterized by conducting trials using Bm86 orthologs on different animal breeds or hosts. Also, the com- Experiment 2 was conducted at the University of Khartoum, bination of different Bm86 orthologs in animal trials could assist Sudan. Seven naïve male camels (Camelus dromedarius) aged 2–2.5 to understand the protective host immune response against tick years old were used in the study. Camels were obtained from a tick infestations. free area geographically localized at the arid zone on the Sahara In this study, vaccination trials were conducted to character- on the northern part of Sudan. Camels were randomly divided into ize the effect of Bm86 vaccination in cattle and camels against H. vaccinated and control groups with four and three animals each, dromedarii and in cattle against A. cajennense immature and adult respectively. Camels were vaccinated with Bm86 while controls ticks. These results expand our knowledge of the effect of Bm86- were not vaccinated. Vaccinated camels received 2 ml intramus- based vaccines in controlling tick infestations and suggest the need cular inoculations of 100 ␮g of recombinant Bm86 in Montanide for Bm86 orthologs or new antigens for the control of multiple tick 888 (GavacTM, Heber Biotec) and equivalent boosters on weeks four species infestations in some regions. and seven. The control group was given phosphate-buffered saline (PBS) solution emulsified in Montanide 888. Ten weeks after the first inoculation, a cotton bag was glued around one ear of each of 2. Materials and methods the camels in both experimental group and 40 laboratory reared unfed H. dromedarii adults (20 females and 20 males two weeks 2.1. Tick species old) were placed in each bag. Ticks were daily checked throughout the feeding period and the fully engorged females that dropped H. dromedarii larvae used in Experiment 1 were originally col- within each cotton bag were collected and immediately weighed. lected from camels in Morocco and maintained at the Faculty of The female ticks were then separately incubated at 28 ◦C and 85% Veterinary Medicine, Utrecht University under controlled condi- relative humidity for egg laying and the weight of individual egg tions at 28 ◦C and 85% relative humidity prior to calf infestation. batches were recorded for each female. Engorged female H. dromedarii were collected at the central live- stock market, Khartoum State, Sudan, and kept feeding on goats 2.4. Experiment 3: Bm86 vaccination against A. cajennense until reared to the subsequent adult stage. Unfed adult ticks were infestations in calves kept in the laboratory prior to the infestation of camels in Experi- ment 2. Experiment 3 was conducted at The Centro Nacional de Par- A colony of A. cajennense was initiated from adult ticks collected asitología, San Antonio de los Banos,˜ Havana, Cuba. Seven naïve from cattle at a farm in Pinar del Río Province of Cuba, in 1999 and calves (Holstein-Friesian) were randomly divided into a vaccinated maintained for 18 months in the Laboratory of the Centro Nacional group containing four cattle (A-46, A-54, V-106 and V-109) and con- de Parasitología, San Antonio de los Banos,˜ Havana, Cuba, on cattle trol group of three calves (R-91, R-94 and R-100). Twelve months (immature and adult ticks). A. cajennense larvae were obtained from old cattle were purchased from tick-free areas. Calves in the vacci- the laboratory colony and maintained at 28 ◦C and >95% relative nated group received 2 ml intramuscular inoculations of 100 ␮gof humidity prior to infesting calves in Experiment 3. All experiments recombinant Bm86 in Montanide 888 (GavacTM, Heber Biotec) and in animals were approved by the Animal Experiments Committees equivalent boosters at weeks 4 and 7. The control group received of the Faculty of Veterinary Medicine at Utrecht University, Khar- PBS with Montanide 888 only. Ten weeks after the first inoculation, toum State, Sudan, and Institute of Veterinary Medicine of Cuba, a total of 1500 A. cajennense larvae (21 days old) were applied to respectively. each calf. These larvae were used to determine vaccination efficacy against the first stage of tick development. The number of 2.2. Experiment 1: Bm86 vaccination against H. dromedarii the engorged nymphs collected was recorded. The effect of Bm86 infestations in cattle on the second stage of A. cajennense life cycle was evaluated by infesting each calf with 75 molted unfed nymphs collected from Experiment 1 was conducted at the Faculty of Veterinary the unvaccinated group. All of the female and male ticks, collected Medicine, Utrecht University, Utrecht, The Netherlands. Four naive from each calf, were matured and used to re-infest the same animal calves (Holstein-Friesian) were randomly divided into two groups to obtain engorged adult ticks. Seven days old adult ticks were used of two animals each. Calves 0813 and 0816 were vaccinated with a to re-infest the cattle. 2 ml subcutaneous inoculation of 100 ␮g of recombinant Bm86 in Montanide 888 (GavacTM, Heber Biotec) and boosted with an equiv- 2.5. ELISA alent dose after four and seven weeks. The other two calves (6844 and 9529) were vaccinated with phosphate-buffered saline (PBS) Sera were collected two weeks after the final vaccination boost solution emulsified in Montanide 888. The immunological response in each experiment to determine the antibody titres to Bm86 using M. Rodríguez-Valle et al. / Vaccine 30 (2012) 3453–3458 3455

Fig. 1. Kinetics of the immune response in camels vaccinated with R. microplus Bm86. () Group vaccinated with Bm86. () Negative control group.

an ELISA [36]. Briefly, 100 ␮l of a solution containing 10 ␮g/ml of compare with data previously obtained in cattle. The anti-Bm86 purified Bm86 was dispensed into each well of a 96-well microtitre- antibody levels were significantly higher (P ≤ 0.05) in all serum plate. The plates were incubated overnight at 4 ◦C and then blocked samples obtained after the last booster dose in the vaccinated group ◦ with 2% skim milk for 1 h at 37 C. Serial two-fold dilutions of bovine (Fig. 1). Vaccinated camels showed a mean OD405 nm of 0.74 ± 0.05 antiserum diluted in phosphate buffer saline (PBS) were added in week 7, while animals in the control group had a mean OD405 nm and the plates were incubated for 2 h at 37 ◦C. The plates were of 0.095 ± 0.05 (Fig. 1). Similar to experiments in cattle and deer washed with PBS–Tween 20 and 100 ␮l of HRP-conjugated goat [33,40–42], antibody titres reached its maximum on week nine anti-bovine IgG per well were added. Plates were incubated for after first immunization with a mean OD405 nm value of 0.93 ± 0.01 1 h at 37 ◦C and then developed with OPD and hydrogen peroxide (Fig. 1). for 15 min under conditions of minimal light exposure. Addition of 2.5 M sulphuric acid stopped the reactions. The optical densi- 3.2. Control of experimental H. dromedarii tick infestations in ties were measured at 492 nm using an Immunoskan-BDSL ELISA cattle and camels reader. For camel sera, a known anti-camel antibody is not com- mercially available, thus protein G labeled with peroxidase diluted Experiments 1 and 2 were conducted to characterize the effect of 1:1000 was used to detect camel antibodies. The plates were then Bm86 vaccination against H. dromedarii infestations in two natural ◦ incubated at 37 C for 1 h. After washing, a substrate buffer (pH hosts, cattle and camels. Vaccination against H. dromedarii imma- 5.0) consisting of ABTS (2,2-zinodi-ethylbenzothiazolinesulfonic ture stages in cattle showed an 89% reduction in the number of acid) tablets (0.5 mg/ml, Sigma) + Na2HPO4 (134 mg/mL) + citric engorged nymphs (Table 1). The reduction of the cumulative tick acid (52.5 mg/ml) was added. After 20 min, the reaction was weight by 98% was statistically significant (P ≤ 0.05) (Table 1). Addi- stopped by adding 50 ␮l of 2.5% sulphuric acid and optical densi- tionally, the analysis of the engorged adult stages of H. dromedarii ties were read at 405 nm using an Immunoskan-BDSL ELISA reader. in vaccinated cattle showed a statistically significant reduction of Data from all experiments were analyzed by the ELISA data analysis 29% (P ≤ 0.05) in the mean weight and 36% reduction of total egg (Ascent; Thermolab System) software. weight per tick (Table 2). The results obtained in Experiment 1 cor- roborated the results obtained by de Vos and colleagues [10] against 2.6. Statistical analysis H. dromedarii in cattle. In camels, Experiment 2 showed a significant prolongation Data collected were processed using general linear model (GLM) of the H. dromedarii tick-feeding period in vaccinated animals procedure using statistical analysis system (SAS version 6.12) with 9.27 ± 0.03 days compared with the control group that package. The SAS was used to perform the analysis of variance had 8.37 ± 0.3 days (P ≤ 0.05). The pre-oviposition period in the (ANOVA) and mean separations were performed according to Ryan–Einot–Gabriel–Welsch Multiple Q test (REGWQ) [37]. Table 1 Effect of R. microplus Bm86 vaccination on H. dromedarii immature tick infestations 3. Results and discussion in cattle.

Experimental group Cumulative number of Cumulative Mean tick 3.1. Characterization of the antibody response in vaccinated engorged nymphs tick weight (g) weight (mg) camels Control Calf no. 6844 362 8.2 22.6 Early experiments have shown that the antibody response Calf no. 9529 587 10.6 18.1 developed by animals vaccinated with recombinant Bm86 anti- Total 949 18.8 gen is essential for the control of R. microplus infestations [18]. Mean ± SD 475 ± 159 9.4 ± 1.7 20.4 ± 3.2 The level of protection against R. microplus is dependent mainly Bm86 Calf no. 0813 90 0.4 18.9 on the anti-Bm86 antibody titers [38,39] and the complement sys- Calf no. 0816 14 0.005 8.8 tem [3]. However, little information exists about the biological Total 104 0.4 factors that may affect the efﬁcacy of the Bm86 vaccination against Mean ± SD 52 ± 54 0.2 ± 0.3 13.9 ± 7.1 * ticks [32,33]. Herein, an ELISA was conducted to measure the anti- Reduction (%) 89 98 32 body levels induced in camels after vaccination with Bm86 and * Statistically signiﬁcant value, p < 0.05. 3456 M. Rodríguez-Valle et al. / Vaccine 30 (2012) 3453–3458

Table 2 Effect of R. microplus Bm86 vaccination on H. dromedarii adult tick infestations in cattle.

Experimental group Cumulative number of Percent engorged ticks Mean tick weight (mg) Percent ticks laying Oviposition (egg engorged ticks (collected/total) eggs weight/tick oviposited) (oviposited/collected) (mg)

Control Calf no. 6844 13 26% (13/50) 0.9 100% (13/13) 0.6 Calf no. 9529 17 34% (17/50) 0.8 100% (17/17) 0.5 Total 30 30% (30/100) 100% (30/30) Mean ± SD 15 ± 3 0.9 ± 0.1 0.6 ± 0.1 Bm86 Calf no. 0813 29 48% (29/60) 0.6 90% (26/29) 0.4 Calf no. 0816 4 36% (4/11) 0.5 100% (4/4) 0.3 Total 33 47% (33/71) 91% (30/33) Mean ± SD 17 ± 18 0.6 ± 0.1 0.4 ± 0.1 Reduction (%) −10 −57 29* 36*

* Statistical signiﬁcant value, p < 0.05.

Table 3 Effect of R. microplus Bm86 vaccination on H. dromedarii adult tick infestations in camels.

Experimental group Ticks applied Cumulative tick number Mean tick weight (mg) Egg laying ticks Egg/tick Hatchability (%)

Control Camel-1C 25 21 0.76 21 0.44 Camel-2C 25 9 0.77 9 0.50 Camel-3C 25 11 0.72 11 0.49 Total 41 41 Mean 0.75 0.48 63.2 ± 0.23a Bm86 Camel-1V 25 13 0.62 13 0.31 Camel-2V 25 12 0.51 11 0.30 Camel-3V 25 5 0.50 4 0.37 Camel-4V 25 14 0.59 14 0.35 Total 44 42 Mean 0.55 0.33 38.8 ± 0.34b Reduction % 26.7* 31.3* 38.6*

Means (±SE) followed by the different letter (a, b) are significantly different at 5% level based on Ryan’s Q test (REGWQ). * p < 0.01. vaccinated group was significantly different with 4.37 ± 0.18 days 3.3. Control of experimental A. cajennense tick infestations in with respect to 3.17 ± 0.09 days for the control group (P ≤ 0.001). cattle In addition, tick oviposition was delayed for the vaccinated group with an average of 17.63 ± 0.21 days when compared to the con- Experiment 3 was conducted to test the effect of Bm86 vac- trol group with 16.66 ± 0.3 days (P ≤ 0.01). Additionally, significant cination against different A. cajennense tick stages. However, reductions (P ≤ 0.05) were shown for the mean weight of engorged vaccination with recombinant Bm86 did not affect A. cajennense tick adult females, the weight of eggs per tick, and the egg hatching rate infestations in cattle. These results were similar to those obtained at 26.7%, 31.3% and 38.6%, respectively (Table 3). The effect of Bm86 by de Vos [10] in cattle infested with A. variegatum, suggesting vaccination against adult H. dromedarii ticks was very similar to the that Bm86 is not effective against Amblyomma spp. tick infestations experiments conducted in cattle. (Tables 4 and 5).

Table 5 Effect of R. microplus Bm86 vaccination on A. cajennense adult tick infestations in Table 4 cattle. Effect of R. microplus Bm86 vaccination on A. cajennense nymph infestations in cattle. Calf no. Ticks applieda Mean tick weight (mg) Egg/tickb Experimental Cumulative number Mean tick Mean tick moulting Control group of engorged nymphs weight (mg) weight (mg) R-91 19F × 23M 0.77 0.45 Control R-94 15F × 26M 0.81 0.49 R-91 42 0.0160 0.007 R-100 24F × 36M 0.77 0.39 R-94 43 0.0190 0.009 Total 2.35 1.33 R-100 60 0.0180 0.008 Mean 0.78 0.44 Total 145 0.0530 0.024 Bm86 Mean 48 0.0177 0.008 A-46 28F × 20M 0.81 0.48 Bm86 A-54 12F × 14M 0.77 0.39 A-46 48 0.0180 0.008 V-106 25F × 22M 0.82 0.44 A-54 26 0.0170 0.008 V-109 11F × 26M 0.74 0.39 V-106 48 0.0180 0.008 Total V-109 38 0.0160 0.008 Mean 0.78** 0.42** Total 160 0.0690 0.032 Reduction % Mean 40* 0.017* 0.008* a Infestation was with all male and female ticks obtained from the same bovine. Reduction % 17 2.36 b Eggs/tick is the egg weight per laying tick. * p > 0.05. ** p > 0.05. M. Rodríguez-Valle et al. / Vaccine 30 (2012) 3453–3458 3457

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