Control of ticks resistant to immunization with Bm86 in cattle vaccinated with the recombinant antigen Bm95 isolated from the cattle tick, Boophilus microplus☆
Introduction
The cattle tick, Boophilus microplus, constitutes a major problem for the cattle industry in tropical and subtropical regions. These ticks cause economic losses due to the diseases they transmit and by directly affecting livestock. The most common method of control involves the use of acaricides, but this has disadvantages, such as the selection of resistant tick populations and harmful effects on the animals, human beings and the environment.
Several approaches have been used to actively immunize bovines against the cattle tick. The first attempts included the use of complex tick extracts [1]. It was not until 1989 that the isolation of the glycoprotein Bm86, the first protective tick antigen, was achieved [2]. When ticks ingest blood from an animal immunized with Bm86, tick gut cells lyse via antibody-mediated mechanisms of host defense, provoking a reduction in tick number, weight, and reproductive capacity [3].
The gene encoding for Bm86 was cloned in Escherichia coli, being expressed in the form of inclusion bodies that conferred immunity to the animals, although to a lower degree than the natural protein [4]. Other expression systems such as Baculovirus and Aspergillus spp. have been used, but expression levels were low [5]. The disadvantages of the expression of Bm86 in those systems were overcome by Rodrı́guez et al. [6] who achieved a high level of expression of the protein in the yeast Pichia pastoris. This recombinant antigen was obtained in a glycosylated and particulated form showing immunogenic and protective properties similar to the natural antigen [7], [8].
In recent years our group has developed an effective vaccine (GavacTM, Heber Biotec S.A., Havana, Cuba), employing a 100 μg/dose of the B. microplus gut Bm86 antigen expressed in P. pastoris [6], [9], [10]. In a number of experiments its effectiveness has been demonstrated against Boophilus ticks [10], [11], [12], [13], especially when appropriately combined with chemical treatments [14], [15], [16]. However, B. microplus isolates showing low susceptibility to vaccination with Bm86 appeared [17], [18], forcing us to look for additional antigens that could be effective against a broader spectrum of tick strains.
In this paper we report on the isolation, cloning and expression, in the yeast P. pastoris, of the antigen Bm95 from strain A of B. microplus. Vaccine formulations containing this antigen proved to be effective against Bm86-sensitive and -resistant B. microplus strains, therefore showing a broader spectrum of action.
Section snippets
Tick strains
The B. microplus strains A (Argentinean Bm86-resistant, CICV-INTA Castelar, [18], [19]) and Camcord (Cuban Bm86-sensitive [18]) were employed in the tick challenge pen trial. Total RNA was extracted from strain A and two Argentinean field isolates (Corrientes Province, Argentina).
Cloning in Escherichia coli and sequencing
Total RNA was extracted from 3 g of B. microplus strain A larvae by the acid phenol extraction method [20]. Total RNA was also extracted from two field isolates (Corrientes Province, Argentina). RNAs were used to
Cloning and sequencing of the Bm95 gene
Total RNA from larvae of the B. microplus strain A, resistant to vaccination with Bm86, was extracted. The cDNA was synthesized from the RNA and was used for the amplification of the Bm86 locus by PCR. The amplification product, named Bm95, was cloned and completely sequenced, obtaining two different sequences, alleles BmA1 and BmA2, differing in four nucleotides that were verified in different clones of two independent amplification reactions (Fig. 2, Table 1). The most striking difference
Discussion
The recombinant Bm86 antigen expressed in the yeast P. pastoris has been successful in controlling tick infestations in a number of controlled and field trials [6], [9], [10], [11], [12], [13], [14], [15]. However, several tick isolates with moderate to low susceptibility to vaccination with Bm86 have been reported [17], [18]. One of these B. microplus isolates, the Argentinian strain A [19], was used to isolate its Bm86-homologue gene Bm95. Since the expression of Bm86 in the yeast P. pastoris
Acknowledgements
The authors wish to thank members of the Mammalian Cell Genetics Division and Technological Development Division (Centro de Ingenierı́a Genética y Biotecnologı́a, Havana, Cuba) and the staff of the Centro Nacional de Parasitologı́a (Havana, Cuba) for fruitful discussions and technical assistance. We would also like to thank Drs Jorge Lamberti (Biogenesis-Syntial S.A., Buenos Aires, Argentina), Alberto Signorini (Programa de Garrapata, SENASA, Buenos Aires, Argentina), Carlos Eddi and Jorge
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Cited by (0)
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The sequence of Bm95 has been deposited in GenBank under the accession number AF150891.
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José C. Garcı́a-Garcı́a, 29434 Crossland Dr., Wesley Chapel, FL 33543, U.S.A.