ORIGINAL PAPER

Efficacy of rBm86 against Rhipicephalus (Boophilus)microplus (IVRI-I line) and Hyalomma anatolicumanatolicum (IVRI-II line) infestations on bovine calves

Binod Kumar & K. Murugan & D. D. Ray &

Srikanta Ghosh

Received: 11 November 2011 /Accepted: 23 February 2012 /Published online: 16 March 2012# Springer-Verlag 2012

Abstract With an aim to evaluate the protective potentialityof rBm86 against Rhipicephalus (Boophilus) microplusIndian Veterinary Research Institute (IVRI)-I line andHyalomma anatolicum anatolicum IVRI-II line infestationson crossbred (Bos indicus×Bos taurus) calves, 20 animalsof 3 months of age were randomly divided in to four equalgroups and maintained in tick-proof conditions. Animals ofgroups 1 and 2 were immunized with 2 ml of rBm86(100 μg)-based vaccine (procured from Revetmex S.A. deC.V, Mexico City, Mexico) thrice at 30 days interval.Animals of groups 3 and 4 were kept as negative controland inoculated with PBS only. Each animal of group 1 and 3was challenged with 7-day-old 50 unfed adults of H. ana-tolicum anatolicum (1:1, male and female), and each animalof groups 2 and 4 was challenged with 6–8-day-old R. (B.)microplus larvae obtained from 50 mg of eggs, on 17th dayof the last immunization. The efficacy of rBm86 against tickinfestations was determined as percentage reduction in num-ber of adults dropped (DT%), engorged body weight(DR%), egg masses (DO%), and immunogen efficacy(E%). The calculated data were 11.8, 10.8, 15.0, and25.1 %, respectively, for DT, DR, DO, and E% against H.anatolicum anatolicum infestation, while in the case of R.(B.) microplus infestation, the corresponding data were 6.4,11.24, 40.7, and 44.5 %, respectively. The results indicatedpartial effectiveness of rBm86 antigen(s) in imparting pro-tection against homologous and heterologous challengeinfestations of Indian ticks. The results indicated identifica-tion of more effective antigen(s) for the development of

vaccine against economically important tick species inIndia.

Introduction

In India, among the 106 reported tick species, Hyalommaanatolicum anatolicum and Rhipicephalus (Boophilus) micro-plus are considered the most damaging and are widely distrib-uted (Ghosh et al. 2007; Ahmed et al. 2007). These tick speciesare of great concern because of their vectorial potential for bothhuman and animal diseases and losses incurred to dairy andmeat industries in terms of growth and production. Currently,ticks control programs are largely dependent upon repeatedapplications of chemical acaricides “on” and “off” the host.This approach, however, has a limited success due to the shorteffective period, harmful side effects, and the development ofacaricide-resistant ticks (Gutteridge 1997; Rosario-Cruz et al.2009; Aguilar-Tipacamú et al. 2008). Therefore, for sustain-able control of ticks, adoption of Integrated Pest Managementinvolving different eco-friendly control methods such as im-munological, biological, exploration of host resistance toticks, herbal, etc. (Willadsen et al. 1989; de la Fuente et al.2007; Ren et al. 2011; Pirali-Kheirabadi et al. 2007; Monteiroet al. 2010; Rees 2004; Wambura et al. 1998; Martinez-Velazquez et al. 2011; Ghosh et al. 2011) is the only option.

In the early 1990s, vaccines inducing immunological pro-tection to vertebrate hosts against tick infestations weredeveloped and commercialized. The commercial vac-cines, Gavac and TickGARDplus, contained the recombinantR. (B.) microplus Bm86 gut antigen. These vaccines reducethe number of engorging female ticks, their weight, andreproductive capacity. Thus, the greatest vaccine effectwas the reduction of larval infestations in subsequentgenerations (Willadsen et al. 1989; Rand et al. 1989;

B. Kumar :K. Murugan :D. D. Ray : S. Ghosh (*)Entomology Laboratory, Parasitology Division, Indian VeterinaryResearch Institute,Izatnagar 243122, Uttar Pradesh, Indiae-mail: sghoshp@yahoo.co.in

Parasitol Res (2012) 111:629–635DOI 10.1007/s00436-012-2879-9

Rodriguez et al. 1994; de la Fuente and Kocan 2003; dela Fuente et al. 2007). Vaccine-controlled field trials incombination with acaricide treatments demonstrated thatan integrated approach resulted in the control of tickinfestations while reducing the use of acaricides (de laFuente et al. 1998, 2007; de la Fuente and Kocan2003). Successful field applications of rBm86 basedvaccine for controlling R. (B.) microplus infestationshave been reported in Australia, Brazil, Cuba, and inmany other Latin American countries (de la Fuente etal. 2007; de la Fuente and Kocan 2003). It was observed thatcontrol of ticks by vaccination has the advantage of beingcost-effective in reducing environmental contamination andpreventing the selection of acaricide-resistant ticks. In addi-tion, these vaccines may also prevent or reduce transmissionof pathogens by reducing tick populations and/or affectingphysiological functions of ticks (de la Fuente et al. 1998,2007; Das et al. 2000, Rodriguez Valle et al. 2004).Moreover, controlled immunization trials have shownBm86-based vaccine having variable efficacy against differentstrains/isolates of R. (B.)microplus (Penichet et al. 1994; de laFuente et al. 1995; Fragoso and Hoshmand 1998; de Vos et al.2001) and cross-protective against other ticks species (de Voset al. 2001; Perez-Perez et al. 2010). The Indian cattle tick,R.(B.) microplus has developed a high level of resistanceagainst commonly used insecticides (Sachin et al. 2011) anda suitable tick vaccine is one of the market demands of theextensive livestock sector of India. Hence, the present inves-tigation was undertaken with an objective to study the efficacyof rBm86 antigen against acaricide susceptible IndianVeterinary Research Institute (IVRI)-I line ofR. (B.)microplusand its cross-protective efficacy against acaricide susceptibleIVRI-II line of H. anatolicum anatolicum.

Materials and methods

Experimental animals

Twenty healthy crossbred calves (Bos indicus×Bos taurus),6–8 months of age were used in this experiment. All calveswere procured from the Livestock Production andManagement section of the Institute just after weaning andmaintained in the tick-proof shed of the Division ofParasitology. These animals had no previous exposure to ticks.The experimental animals were maintained as per the guide-lines of Committee for the Purpose of Control and Supervisionon Experiments on Animals, a statutory Indian body.

Ticks

The homogenous acaricide susceptible Babesia bigemina-free R. (B.) microplus IVRI-I line and acaricide susceptible

Theileria annulata-free H. anatolicum anatolicum IVRI-IIline were maintained in the Entomology Laboratory of theDivision of Parasitology as per the methods of Ghoshand Azhahianambi (2007). Sterile cotton ear bags wereused for feeding of ticks on animals. For raising goodnumber of tick stages and to avoid stress on animals,six to eight rabbits and five to six calves were main-tained simultaneously.

R. (B.) microplus

Healthy calves of 6–7 months old were used for feeding ofticks. TheB. bigemina free status of the calves was ascertainedby periodical examination of Giemsa-stained blood smears.Usually two to three calves were used for each feeding cycle.Fully engorged adult female ticks were kept in desiccatorswhere 85 % relative humidity (RH) was maintained andincubated at 28°C for oviposition. After completion of ovi-position, the dead female ticks were removed from theglass tubes in order to avoid fungal growth. The freshlylaid eggs were kept at 28°C with 85 % RH. The freshlyhatched larvae were kept unfed for 6–8 days. Theselarvae were used for challenge study.

H. anatolicum anatolicum

Healthy New Zealand white rabbits of 9 months to 1 year oldand 1.5–2 kg in weight were used for feeding of larvae of H.anatolicum anatolicum. Before releasing ticks on animals, theear pinnas were shaved without damaging the skin. Afterrelease of tick larvae, the animals were checked daily twice,in the morning and in the evening, to avoid the loss of tickstages. After feeding, the engorged larvae remained on rabbitsto moult into unfed nymphs, which then attach and engorge.The engorged nymphs were collected and cleaned beforeplacing in tick rearing glass vials. The tubes containingengorged nymphs were kept at 28°C and in 85 % RH formoulting. The freshly hatched adults were kept unfedfor 7 days. These 7-day-old unfed adult ticks were usedto study the vaccine efficacy. Usually, the adults werereleased in the morning hours of the day and wereclosely observed for 24 h. During the initial stage oftick attachment (up to 12 h of release), the animalssuffered from irritation and try to remove the tick feed-ing bag. The ear bags were checked daily, the fed adultswere collected, cleaned, weighed, labeled, and kept sin-gly in the glass tubes at 28°C and 85 % RH.

Vaccine

The rBm86-based vaccine manufactured by Revetmex S.A.de C.V, Mexico City, Mexico had a concentration of rBm86at 50 μg/ml.

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Cattle immunizations and tick infestations

For immunization, animals were divided randomly into fourgroups comprising of five animals in each group. Animalsof groups 1 and 2 were immunized with 2 ml of rBm86-based vaccine, whereas animals of groups 3 and 4 were keptas negative control [inoculated with phosphate-buffered saline(PBS) only] on the 0, 30, and 60th day. The immunization wascarried out by deep intramuscular inoculation in the glutealmuscle.

Unfed 7-day-old adults of H. anatolicum anatolicum and6–8-day-old larvae of R. (B.) microplus were used for chal-lenge study. On the 17th day of the second booster, animalswere challenged as follows: each animal of groups 1 and 3was challenged with 50 unfed adults (male and female, 1:1ratio) of H. anatolicum anatolicum. Animals of groups 2and 4 were challenged with larvae emanated from 50-mgeggs of R. (B.) microplus. The tick stages were released onear pinna using ear bag method (Ghosh and Azhahianambi2007). The engorged adults of H. anatolicum anatolicumand adults of R. (B.) microplus were collected by checkingthe ear bag daily and the efficacy of vaccine formulationswas evaluated (Ghosh et al. 2005).

DT% ¼ 100 1� NTV=NTCð Þ;Where DT% is the percentage reduction of females, NTV

is the number of females dropped from the immunizedgroup of animals, and NTC is the number of femalesdropped from the control group of animals.

DO %ð Þ ¼ 100 1� PATV=PATCð ÞWhere, DO (%) is the percentage reduction of mean

weight of eggs, PATV is the mean weight of eggs of femalesfed on immunized group of animals, and PATC is the meanweight of eggs of females fed on control group of animals.

DR %ð Þ ¼ 100 1� PMTV=PMTCð ÞWhere, DR (%) is the percentage reduction of mean

weight of an adult female, PMTV is the mean weight of an

adult female dropped from the immunized group of animals,and PMTC is the mean weight of an adult female droppedfrom the control group of animals.

E %ð Þ ¼ 100 1� CRT� CROð Þ½ �Where, E (%) is the efficacy of immunogen. CRO is the

reduction in egg-laying capacity (PATV/PATC), and CRT isthe reduction in the number of adult females (NTV/NTC).

Statistical analysis

The analysis of variance was used for comparing the entomo-logical data after challenge infestations among the differentgroups of the experimental calves. Significance at 5 % level(P<0.05) was used to define differences in different parameters.

Results

Effect of immunization on feeding, moulting,and reproductive performances of challenged ticks

The calves were clinically normal following inoculations ofrBm86 antigen during the pre-challenge period of 75 days.There were no local reactions at the site of inoculation. Thecomparative efficacy of rBm86-based vaccine against chal-lenged infestations of R. (B.) microplus and H. anatolicumanatolicum on experimental calves was presented in Fig. 1.The mean difference in number of ticks dropped, engorge-ment weight, and the mean egg masses (milligrams) laid bythe ticks dropped from calves of different groups werecompared and presented in Table 1.

R. (B.) microplus

The larvae start feeding on the animals about 48 h of releaseand it continues for the next 16–17 days before dropped asengorged adults. No significant differences in the number ofdropped females were recorded between the two groups.

6.4

11.24

40.744.5

11.8 10.815

25.1

DT% DR% DO% E%

R. (B.) microplus H. a. anatolicumFig. 1 Comparative efficacy ofBm86-based vaccine againstchallenge infestation of R. (B.)microplus and H. anatolicumanatolicum

Parasitol Res (2012) 111:629–635 631

However, significant differences in the engorgement weightand egg masses were recorded when the corresponding datawere compared between the ticks fed on groups 2 and 4 (p<0.05; p<0.001). A small number of engorged ticks collectedfrom animals of group 2 were black in color and normalmorphology was distorted (Fig. 2). The DT%, DO%, andDR% of ticks fed on group 2 animals were determined as6.4, 40.7, and 11.24 %, respectively. The E% of the antigenwas determined as 44.5 %.

H. anatolicum anatolicum

The unfed adult ticks start feeding about 48 h of release onthe experimental animals. Analysis of entomological datashows there were no significant differences in the meannumber of ticks dropped from control and immunized group

of animals. The data were insignificant when comparing theengorgement weight of ticks fed on animals of group 1 and3. When the mean egg masses (milligrams) laid by the ticksthat dropped from the calves of these two groups werecompared, the data were found significant at p<0.05 level.Figure 3 showed the effect of vaccination on the adult tickswhere few were not able to feed properly on vaccinatedcalves. The direct effect of immunization with rBm86 onnumber of females was 11.8 %. The other entomologicalparameters, viz., DO%, DR%, and E% were 15.0, 10.8, and25.1 %, respectively, compared to control animals.

Discussion

R. (B.) microplus and H. anatolicum anatolicum are the twomajor tick species of Indian cattle which cause great loss to

Table 1 Effect of cattle vaccination with rBm86 on H. anatolicum anatolicum adults (IVRI-II line) and R. (B.) microplus (IVRI-I line) infestations

Vaccine efficacy parametersa Experimental groupsb

Vaccinated with rBm86 PBS (negative control)

Challenged with R. (B.)microplus

Challenged with H. anatolicumanatolicum

Challenged with R. (B.)microplus

Challenged with H. anatolicumanatolicum

DT% 6.4 (116.3±5.43) 11.8 (15±3.08) (124.3±8.32) (17±2.54)

DO% 40.7 (29.07±1.6)* 15.0 (223.3±11.5)* (49.03±1.5) (262.9±12.6)

DR% 11.24 (92.4±2.3)* 10.8 (385.0±16.4) (104.1±1.6) (431.5±18.5)

E%c 44.5 25.1

*p<0.05a The percent reduction was calculated with respect to the control group: DT, percent reduction in tick infestation; DR, percent reduction in tickweight; DO, percent reduction in oviposition. In parenthesis are shown the average±SE for adult female tick number, tick weight (milligrams),oviposition [egg weight (milligrams)/tick] and were compared by ANOVAwith unequal variance between vaccinated and control groups (*p<0.05)b Cattle were randomly assigned to experimental groups (N05), vaccinated, and challenged with H. anatolicum anatolicum unfed adult and R. (B.)microplus larvaec Vaccine efficacy (E) was calculated as 100[1−(CRT×CRO)], where CRT and CRO are the reduction in the number of adult female ticks andoviposition as compared to the control group, respectively

Fig. 3 Engorged H. anatolicum anatolicum dropped from rBm86immunized and control groups of animals. There were few tickscollected from immunized animals showing incomplete feeding

Fig. 2 Engorged R. (B.) microplus dropped from rBm86 immunizedand control groups of animals. Ticks collected from immunized animalshowing abnormal morphology as loss of normal body texture andcolor

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Indian dairy and leather industry (Devendra 1995; Biswas2003). The successful application of Bm86-based vaccine inAustralia and Latin American countries created great curi-osity amongst researchers from different parts of world. Theefficacy of Bm86-based vaccine against different strains ofR. (B.) microplus and breeds of cattle ranged from 51 to91 % with the same dose of vaccine (Penichet et al. 1994;Floyd et al. 1995; Rodriguez et al. 1995; de la Fuente et al.2007). In the present vaccination trial using the same dose ofvaccine, a 44.5 % efficacy against R. (B.) microplus wasrecorded. The major contributor of vaccine efficacy wasreduction in egg masses which might be due to antibody-mediated gut damage and resultant reduction in bioticpotential of ticks. After homologous challenge of calves, nosignificant reduction was observed in the number of ticksdropped, while there was a significant reduction in bodyweight and egg masses laid by the engorged ticks. It isnoteworthy that physical damage observed in adults wasapparent in few ticks, suggesting a rapid and direct damageon the feeding ticks besides the effect on successive gener-ations following vaccination. However, Almazán et al. (2010)had observed that the efficacy (E%060) of Bm86-based vac-cine against homologous challenge infestation on calves wasmainly due to reduction in number of engorged ticks (DT%051).

As reported by many workers (de la Fuente and Kocan2003, 2006; Willadsen 2004; Ghosh et al. 2006) from thecommercial point of view, the ideal tick vaccine should becross-protective. Early experiment with Bm86-based vac-cines demonstrated cross-protection against Rhipicephalus(Boophilus) annulatus and Rhipicephalus (Boophilus)decoloratus infestations and conferred partial protectionagainst Hyalomma and Rhipicephalus spp. (de la Fuente etal. 2000; Fragoso and Hoshmand 1998; de Vos et al. 2001;Perez-Perez et al. 2010). However, immunization withBm86 failed to protect animals against Amblyomma spp.(de Vos et al. 2001) and against some geographical strainsof R. (B.) microplus (Garcia-Garcia et al. 1999, 2000). Theseresults supported the use of Bm86 vaccine in regions whereRhipicephalus (Boophilus) spp. coexist with other tickgenera, as frequently occurs in regions of America, Africa,and Asia. However, these results also suggested the need fordiscovery of more conserved tick-protective antigens thatcould be used to control infestations by multiple tick spp. inwide geographical areas. Recently, the ortholog of the tickprotective antigens, subolesin, was identified in Aedes albo-pictus and found to have conserved epitopes in ticks andmosquitoes (Canales et al. 2009). The vaccine efficacy ofrecombinant subolesin was encouraging against bothhomologous and heterologous challenge infestations(Almazán et al. 2010; Carreón et al. 2012) and createsa hope for the development of a broad-spectrum vaccineagainst different tick species.

In the present experiment, the cross-protective efficacy ofBm86 is evaluated against H. anatolicum anatolicum, oneof the most predominant tick species of India. The efficacyof vaccine was not satisfactory. The efficacy data showed aninsignificant reduction in number and body weight ofengorged ticks dropped from vaccinated animals. But thedata were significant (p<0.05) for reduction in egg masses.The E% was estimated as 25.1 %, and the value was notsatisfactory. Although de Vos et al. (2001) reported a sig-nificant efficacy of Bm86-based vaccine against Ludhianaisolates of H. anatolicum, the result was not repeated in thepresent experiments.

The result indicated partial effectiveness of Bm86 antigenin imparting protective response against homologous chal-lenge infestation of R. (B.) microplus IVRI-I line. Therewere insignificant differences in DT% and DR%, but asignificant difference in DO% was recorded. The majorcontributor to the higher efficacy of vaccine was reducedoviposition (DO%) in homologous challenge infestations.The effect of vaccine on oviposition (DO%) of H. anatoli-cum anatolicum was less, which may be due to lesserfeeding period (5–6 days) as well as the source of heterol-ogous antigen.

Previously, we evaluated the efficacy of Bm86 homo-logue of H. anatolicum anatolicum, rHaa86, against R. (B.)microplus and the recorded efficacy was 36.5 % (Kumar etal. 2012). The present experiment confirms our earlier con-tention that a vaccine developed from a homologous antigenhas better efficacy than the vaccine from a heterologous one.

Acknowledgments Sincere thanks are due to the Department ofBiotechnology, Government of India for the funding. Senior author ishighly thankful to the Indian Council of Agriculture Research (ICAR)for providing Senior Research fellowship. The technical support pro-vided by the laboratory staff (Mr. Laxmi Lal and Naresh Kumar) of theentomology laboratory is highly acknowledged.

Conflict of interest There is no conflict of interest.

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