Efficacy of Rhipicephalus (Boophilus) microplus ...

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 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 b

a r t i c l e

i n f o

Article history: Received 5 January 2012 Received in revised form 13 February 2012 Accepted 8 March 2012 Available online 22 March 2012 Keywords: Rhipicephalus microplus Hyalomma dromedarii Amblyomma cajennense Tick Vaccine Bm86 Cattle Camels

a b s t r a c t The recombinant Bm86-based tick vaccines have shown their efficacy for the control of cattle ticks, Rhipicephalus (Boophilus) microplus and R. annulatus infestations. However, cattle ticks often co-exist with multi-host ticks such as Hyalomma and Amblyomma species, thus requiring the control of multiple tick infestations for cattle and other hosts. Vaccination trials using a R. microplus recombinant Bm86based 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 H. dromedarii nymphs engorging on vaccinated cattle, and a further 32% reduction in the weight of the surviving adult ticks. In vaccinated camels, a reduction of 27% and 31% of tick engorgement and egg mass weight, respectively was shown, while egg hatching was reduced by 39%. However, cattle vaccination with Bm86 did not have an effect on A. cajennense tick infestations. These results showed that Bm86 vaccines are effective against R. microplus and other tick species but improved vaccines containing new antigens are required to control multiple tick infestations. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Bm86 is a membrane-bound glycoprotein on the surface of R. microplus gut cells [1]. In 1995, the antigen Bm86 was re-cloned from an Argentinean strain of R. microplus and designated as Bm95 [2]. Antibodies produced in vaccinated cattle bind to Bm86 in the tick gut and are thought to cause damage mainly by the complement pathway, but this mechanism is still poorly understood [3,4]. The result of Bm86 vaccination is the reduction in the number and weight of engorged ticks, the egg laying capacity and the egg fertility, which ultimately reduces tick populations and the prevalence of tick-borne pathogens [5–8]. Bm86 is present in dif-

∗ Corresponding author at: Queensland Alliance for Agriculture & Food Innovation (QAAFI), Queensland Biosciences Precinct, The University of Queensland, DEEDI, GPO Box 6097, St Lucia, Qld 4067, Australia. Tel.: +61 0 7 3255 4529. E-mail address: [email protected] (M. Rodríguez-Valle). 1 306 Carmody Rd (Building 80 Loading Dock, off Services Road), St. Lucia, Qld 4072, Australia. 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2012.03.020

ferent cattle tick strains [9] and in other tick species [10–14] as Bm86 orthologs. During 1993–1997, two vaccines using recombinant Bm86 were registered for use in Latin American countries (GavacTM ) and Australia (TickGARDTM ) [6,15–17] and had similar efficacy for the control of cattle tick infestations [18]. These tick vaccines constitute the only example of commercial vaccines for the control of ectoparasites and led the way for research on the development of vaccines for arthropod vectors of pathogens that affect human and animal health [18]. In many regions, R. microplus shares its habitat with other Rhipicephalus tick species such as R. annulatus, R. decoloratus and R. geigyi 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 the North, North East, East and Sahara-Sahel regions of Africa that occasionally uses three hosts [19,22]. Camels are the principal host of the H. dromedarii adults with some records also showing that cattle and goats are also susceptible [23]. The immature stages can parasitize rodents, leporids, hedgehogs and birds [23]. In addition,

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H. dromedarii is a vector of bacteria [24], protozoa [25], rickettsia [26], and viruses [27]. In the Americas, Rocky Mountain spotted fever caused by Rickettsia rickettsii is transmitted by A. cajennese, Dermacentor andersoni, D. variablis and R. sanguineus [28]. The Rickettsia amblyommii is transmitted by A. cajennese in Brazil [29]. Recombinant Bm86 continues to be the most effective antigen against tick infestations. The protective efficacy of vaccination with Bm86-based vaccines has been shown an effect on the control of tick infestations by Hyalomma anatolicum anatolicum, R. annulatus, R. decoloratus and H. dromedarii [10,30–32]. Close to 100% control of R. annulatus infestations has been observed using Bm86 cattle vaccination in different trials [10,30,31,33]. In contrast, Bm86 has shown partial or no protection against some R. microplus strains from different geographic regions and other tick species [10,18,30,34,35]. The effect of Bm86 vaccination against different tick species can be further characterized by conducting trials using Bm86 orthologs on different animal breeds or hosts. Also, the combination of different Bm86 orthologs in animal trials could assist to understand the protective host immune response against tick infestations. In this study, vaccination trials were conducted to characterize the effect of Bm86 vaccination in cattle and camels against H. dromedarii and in cattle against A. cajennense immature and adult ticks. These results expand our knowledge of the effect of Bm86based vaccines in controlling tick infestations and suggest the need for Bm86 orthologs or new antigens for the control of multiple tick species infestations in some regions. 2. Materials and methods 2.1. Tick species H. dromedarii larvae used in Experiment 1 were originally collected from camels in Morocco and maintained at the Faculty of Veterinary Medicine, Utrecht University under controlled conditions at 28 ◦ C and 85% relative humidity prior to calf infestation. Engorged female H. dromedarii were collected at the central livestock market, Khartoum State, Sudan, and kept feeding on goats until reared to the subsequent adult stage. Unfed adult ticks were kept in the laboratory prior to the infestation of camels in Experiment 2. A colony of A. cajennense was initiated from adult ticks collected from cattle at a farm in Pinar del Río Province of Cuba, in 1999 and maintained for 18 months in the Laboratory of the Centro Nacional ˜ de Parasitología, San Antonio de los Banos, Havana, Cuba, on cattle (immature and adult ticks). A. cajennense larvae were obtained from the laboratory colony and maintained at 28 ◦ C and >95% relative humidity prior to infesting calves in Experiment 3. All experiments in animals were approved by the Animal Experiments Committees of the Faculty of Veterinary Medicine at Utrecht University, Khartoum State, Sudan, and Institute of Veterinary Medicine of Cuba, respectively. 2.2. Experiment 1: Bm86 vaccination against H. dromedarii infestations in cattle Experiment 1 was conducted at the Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Four naive calves (Holstein-Friesian) were randomly divided into two groups of two animals each. Calves 0813 and 0816 were vaccinated with a 2 ml subcutaneous inoculation of 100 ␮g of recombinant Bm86 in Montanide 888 (GavacTM , Heber Biotec) and boosted with an equivalent dose after four and seven weeks. The other two calves (6844 and 9529) were vaccinated with phosphate-buffered saline (PBS) solution emulsified in Montanide 888. The immunological response

of vaccinated and control cattle was determined by ELISA [36]. The titres of anti-Bm86 IgG were very high in the vaccinated group after the last immunization with Bm86, as it is reported in the literature [17]. Ten weeks after vaccination, a cage containing approximately 1500 H. dromedarii larvae (three months old) was glued onto the back of each calf. Engorged nymphs were collected separately from each calf and reapplied to the same calves in a similar way. Moulted unfed adult ticks applied were five months old. Tick infestations were monitored daily. Engorged adults tick were collected, counted and weighed. Female ticks were incubated at 28 ◦ C and 85% relative humidity for egg laying and weight. 2.3. Experiment 2: Bm86 vaccination against H. dromedarii infestations in camels Experiment 2 was conducted at the University of Khartoum, Sudan. Seven naïve male camels (Camelus dromedarius) aged 2–2.5 years old were used in the study. Camels were obtained from a tick free area geographically localized at the arid zone on the Sahara on the northern part of Sudan. Camels were randomly divided into vaccinated and control groups with four and three animals each, respectively. Camels were vaccinated with Bm86 while controls were not vaccinated. Vaccinated camels received 2 ml intramuscular inoculations of 100 ␮g of recombinant Bm86 in Montanide 888 (GavacTM , Heber Biotec) and equivalent boosters on weeks four 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 the camels in both experimental group and 40 laboratory reared unfed H. dromedarii adults (20 females and 20 males two weeks old) were placed in each bag. Ticks were daily checked throughout the feeding period and the fully engorged females that dropped within each cotton bag were collected and immediately weighed. The female ticks were then separately incubated at 28 ◦ C and 85% relative humidity for egg laying and the weight of individual egg batches were recorded for each female. 2.4. Experiment 3: Bm86 vaccination against A. cajennense infestations in calves Experiment 3 was conducted at The Centro Nacional de Par˜ asitología, San Antonio de los Banos, Havana, Cuba. Seven naïve calves (Holstein-Friesian) were randomly divided into a vaccinated group containing four cattle (A-46, A-54, V-106 and V-109) and control group of three calves (R-91, R-94 and R-100). Twelve months old cattle were purchased from tick-free areas. Calves in the vaccinated group received 2 ml intramuscular inoculations of 100 ␮g of recombinant Bm86 in Montanide 888 (GavacTM , Heber Biotec) and equivalent boosters at weeks 4 and 7. The control group received PBS with Montanide 888 only. Ten weeks after the first inoculation, a total of 1500 A. cajennense larvae (21 days old) were applied to each calf. These larvae were used to determine vaccination efficacy against the first stage of tick development. The number of the engorged nymphs collected was recorded. The effect of Bm86 on the second stage of A. cajennense life cycle was evaluated by infesting each calf with 75 molted unfed nymphs collected from the unvaccinated group. All of the female and male ticks, collected from each calf, were matured and used to re-infest the same animal to obtain engorged adult ticks. Seven days old adult ticks were used to re-infest the cattle. 2.5. ELISA Sera were collected two weeks after the final vaccination boost in each experiment to determine the antibody titres to Bm86 using

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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 purified Bm86 was dispensed into each well of a 96-well microtitreplate. The plates were incubated overnight at 4 ◦ C and then blocked with 2% skim milk for 1 h at 37 ◦ C. Serial two-fold dilutions of bovine antiserum diluted in phosphate buffer saline (PBS) were added and the plates were incubated for 2 h at 37 ◦ C. The plates were washed with PBS–Tween 20 and 100 ␮l of HRP-conjugated goat anti-bovine IgG per well were added. Plates were incubated for 1 h at 37 ◦ C and then developed with OPD and hydrogen peroxide for 15 min under conditions of minimal light exposure. Addition of 2.5 M sulphuric acid stopped the reactions. The optical densities were measured at 492 nm using an Immunoskan-BDSL ELISA reader. For camel sera, a known anti-camel antibody is not commercially available, thus protein G labeled with peroxidase diluted 1:1000 was used to detect camel antibodies. The plates were then incubated at 37 ◦ C for 1 h. After washing, a substrate buffer (pH 5.0) consisting of ABTS (2,2-zinodi-ethylbenzothiazolinesulfonic acid) tablets (0.5 mg/ml, Sigma) + Na2 HPO4 (134 mg/mL) + citric acid (52.5 mg/ml) was added. After 20 min, the reaction was stopped by adding 50 ␮l of 2.5% sulphuric acid and optical densities were read at 405 nm using an Immunoskan-BDSL ELISA reader. Data from all experiments were analyzed by the ELISA data analysis (Ascent; Thermolab System) software. 2.6. Statistical analysis Data collected were processed using general linear model (GLM) procedure using statistical analysis system (SAS version 6.12) package. The SAS was used to perform the analysis of variance (ANOVA) and mean separations were performed according to Ryan–Einot–Gabriel–Welsch Multiple Q test (REGWQ) [37]. 3. Results and discussion 3.1. Characterization of the antibody response in vaccinated camels Early experiments have shown that the antibody response developed by animals vaccinated with recombinant Bm86 antigen is essential for the control of R. microplus infestations [18]. The level of protection against R. microplus is dependent mainly on the anti-Bm86 antibody titers [38,39] and the complement system [3]. However, little information exists about the biological factors that may affect the efficacy of the Bm86 vaccination against ticks [32,33]. Herein, an ELISA was conducted to measure the antibody levels induced in camels after vaccination with Bm86 and

compare with data previously obtained in cattle. The anti-Bm86 antibody levels were significantly higher (P ≤ 0.05) in all serum samples obtained after the last booster dose in the vaccinated group (Fig. 1). Vaccinated camels showed a mean OD405 nm of 0.74 ± 0.05 in week 7, while animals in the control group had a mean OD405 nm of 0.095 ± 0.05 (Fig. 1). Similar to experiments in cattle and deer [33,40–42], antibody titres reached its maximum on week nine after first immunization with a mean OD405 nm value of 0.93 ± 0.01 (Fig. 1). 3.2. Control of experimental H. dromedarii tick infestations in cattle and camels Experiments 1 and 2 were conducted to characterize the effect of Bm86 vaccination against H. dromedarii infestations in two natural hosts, cattle and camels. Vaccination against H. dromedarii immature stages in cattle showed an 89% reduction in the number of engorged nymphs (Table 1). The reduction of the cumulative tick weight by 98% was statistically significant (P ≤ 0.05) (Table 1). Additionally, the analysis of the engorged adult stages of H. dromedarii in vaccinated cattle showed a statistically significant reduction of 29% (P ≤ 0.05) in the mean weight and 36% reduction of total egg weight per tick (Table 2). The results obtained in Experiment 1 corroborated the results obtained by de Vos and colleagues [10] against H. dromedarii in cattle. In camels, Experiment 2 showed a significant prolongation of the H. dromedarii tick-feeding period in vaccinated animals with 9.27 ± 0.03 days compared with the control group that had 8.37 ± 0.3 days (P ≤ 0.05). The pre-oviposition period in the Table 1 Effect of R. microplus Bm86 vaccination on H. dromedarii immature tick infestations in cattle. Experimental group Control Calf no. 6844 Calf no. 9529 Total Mean ± SD Bm86 Calf no. 0813 Calf no. 0816 Total Mean ± SD Reduction (%) *

Cumulative number of engorged nymphs

Cumulative tick weight (g)

Mean tick weight (mg)

362 587 949 475 ± 159

8.2 10.6 18.8 9.4 ± 1.7

22.6 18.1

90 14 104 52 ± 54 89

0.4 0.005 0.4 0.2 ± 0.3 98*

18.9 8.8

Statistically significant value, p < 0.05.

20.4 ± 3.2

13.9 ± 7.1 32

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

Control Calf no. 6844 Calf no. 9529 Total Mean ± SD Bm86 Calf no. 0813 Calf no. 0816 Total Mean ± SD Reduction (%) *

Percent engorged ticks (collected/total)

Cumulative number of engorged ticks

13 17 30 15 ± 3

26% (13/50) 34% (17/50) 30% (30/100)

Mean tick weight (mg)

0.9 0.8

Percent ticks laying eggs (oviposited/collected)

Oviposition (egg weight/tick oviposited) (mg)

100% (13/13) 100% (17/17) 100% (30/30)

0.6 0.5

0.9 ± 0.1

29 4 33 17 ± 18 −10

48% (29/60) 36% (4/11) 47% (33/71) −57

0.6 0.5

0.6 ± 0.1 90% (26/29) 100% (4/4) 91% (30/33)

0.4 0.3

0.6 ± 0.1 29*

0.4 ± 0.1 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 Control Camel-1C Camel-2C Camel-3C Total Mean Bm86 Camel-1V Camel-2V Camel-3V Camel-4V Total Mean Reduction %

Ticks applied

Cumulative tick number

25 25 25

21 9 11 41

Mean tick weight (mg) 0.76 0.77 0.72

Egg laying ticks

Egg/tick

21 9 11 41

0.44 0.50 0.49

0.75 25 25 25 25

13 12 5 14 44

0.48

0.62 0.51 0.50 0.59

Hatchability (%)

13 11 4 14 42

63.2 ± 0.23a

0.31 0.30 0.37 0.35

0.55 26.7*

0.33 31.3*

38.8 ± 0.34b 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 with respect to 3.17 ± 0.09 days for the control group (P ≤ 0.001). In addition, tick oviposition was delayed for the vaccinated group with an average of 17.63 ± 0.21 days when compared to the control group with 16.66 ± 0.3 days (P ≤ 0.01). Additionally, significant reductions (P ≤ 0.05) were shown for the mean weight of engorged adult females, the weight of eggs per tick, and the egg hatching rate at 26.7%, 31.3% and 38.6%, respectively (Table 3). The effect of Bm86 vaccination against adult H. dromedarii ticks was very similar to the experiments conducted in cattle.

Table 4 Effect of R. microplus Bm86 vaccination on A. cajennense nymph infestations in cattle. Experimental group Control R-91 R-94 R-100 Total Mean Bm86 A-46 A-54 V-106 V-109 Total Mean Reduction % *

p > 0.05.

Cumulative number of engorged nymphs

Mean tick weight (mg)

Mean tick moulting weight (mg)

42 43 60 145 48

0.0160 0.0190 0.0180 0.0530 0.0177

0.007 0.009 0.008 0.024 0.008

48 26 48 38 160 40* 17

0.0180 0.0170 0.0180 0.0160 0.0690 0.017* 2.36

0.008 0.008 0.008 0.008 0.032 0.008*

3.3. Control of experimental A. cajennense tick infestations in cattle Experiment 3 was conducted to test the effect of Bm86 vaccination against different A. cajennense tick stages. However, vaccination with recombinant Bm86 did not affect A. cajennense tick infestations in cattle. These results were similar to those obtained by de Vos [10] in cattle infested with A. variegatum, suggesting that Bm86 is not effective against Amblyomma spp. tick infestations (Tables 4 and 5).

Table 5 Effect of R. microplus Bm86 vaccination on A. cajennense adult tick infestations in cattle. Calf no. Control R-91 R-94 R-100 Total Mean Bm86 A-46 A-54 V-106 V-109 Total Mean Reduction % a b **

Ticks applieda

Mean tick weight (mg)

Egg/tickb

19F × 23M 15F × 26M 24F × 36M

0.77 0.81 0.77 2.35 0.78

0.45 0.49 0.39 1.33 0.44

28F × 20M 12F × 14M 25F × 22M 11F × 26M

0.81 0.77 0.82 0.74

0.48 0.39 0.44 0.39

0.78**

0.42**

Infestation was with all male and female ticks obtained from the same bovine. Eggs/tick is the egg weight per laying tick. p > 0.05.

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4. General discussion and conclusions Important advances have been made in the purification and identification of antigens able to elicit a protective immunological response against tick infestation based on the advances of genomics and RNA interference tools for the study and genetic manipulation of ticks [43]. However, Bm86 remains the most efficacious among the candidate tick antigens described to date [8,18,30,38,44]. The potential utility of Bm86 and its orthologs for the control of other tick species have been described [10,14,30–32,45]. Additionally, Bm86 orthologs have been sequenced and cloned from other tick species such as Hyalomma anatolicum [10], Hyalomma scupense (syn. H detritum) [11] and Haemaphysalis longicornis [46]. Sequence variation has also been reported in the Bm86 locus of R. microplus [34,47,48]. These studies have shown high variability in Bm86 DNA and amino acid sequences within R. microplus strains and high homology with other tick species such as R. decoloratus [14], R. annulatus [33,34], H. longicornis [46], H. anatolicum anatolicum [10], and Hyalomma scupense (syn. H detritum) [11]. The variation of Bm86 ortholog gene sequences among different R. microplus strains and between tick species may not be sufficient to explain the differences in vaccine susceptibility found within R. microplus strains [30,38,47] and against other tick species such as R. appendiculatus [10] and R. annulatus [33]. Tick biological factors such as clearance of the host anti-Bm86 IgGs by tick immunoglobulin binding proteins [49] or gut protease activity and/or the inhibition of complement activity [3,50] may be important to explain Bm86 vaccine efficacy against different tick species. In summary, the results reported here confirmed the efficacy of the vaccination with R. microplus recombinant Bm86 for the control of H. dromedarii infestations in cattle and camels. Field trials with various breeds of cattle and camels are needed to confirm the reported laboratory results. However, there is enough evidence to suggest that immunization of cattle and camels with the Bm86 vaccine represents an effective alternative for controlling H. dromedarii tick infestations. Cloning and characterization of Bm86 orthologs in Hyalomma tick spp. will provide additional tools for the control of these tick species. Additionally, in regions where R. annnulatus, R. microplus, H. anatolicum and/or H. dromedarii coexist would be ideal locations to undertake field trials with vaccine formulations containing combinations of Bm86 orthologs. However, cattle vaccination with Bm86 did not have an effect on A. cajennense tick infestations. These results showed that Bm86 vaccines are effective against R. microplus and other tick species but improved vaccines containing new antigens are required to control multiple tick infestations. Conflicts of interests Authors have no conflicts of interests. Acknowledgments This work has been facilitated through The Integrated Consortium on Ticks and Tick-borne Diseases (ICTTD-3) financed by the International Cooperation Program of the European Union through Coordination Action Project No. 510561. The authors wish to thank all veterinary technicians who contributed to this work and to Ala Lew-Tabor and Wayne Jorgensen (Queensland Alliance for Agriculture & Food Innovation, The University of Queensland, QLD, Australia) for their comments on this paper.

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