Joao Carlos Gonzales

Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus

An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.

IMMUNIZATION OF BOVINES WITH AN ASPARTIC PROTEINASE PRECURSOR ISOLATED FROM BOOPHILUS MICROPLUS EGGS

The capacity of the Boophilus Yolk pro-Cathepsin (BYC) to induce a protective immune response in cattle against Boophilus microplus infestation was tested by vaccination experiments and by inoculation of monoclonal antibody (MAb) against BYC into fully engorged tick females. In immunization experiments the measurement of various biological parameters demonstrated a partial protection against B. microplus. A continuous decrease in the levels of specific antibodies was observed over 11 months when six bovines were maintained in field conditions. The inoculation of the MAb into tick females produced a dose-dependent decrease in oviposition and survival of the ectoparasite compared to the control.

Therapeutic and persistent efficacy of doramectin against Boophilus microplus in cattle

One therapeutic and one persistent efficacy study were conducted in Brazil to evaluate doramectin at a dose rate of 200 micrograms kg-1 against induced infestations of the single host tick, Boophilus microplus. Doramectin was highly effective in eliminating established tick populations from cattle and also in preventing infestation by the parasite. In the therapeutic study, 12 calves were infested three times a week along the dorsal line with 2500 recently hatched larvae, for a total of 11 times before treatment. Animals were allocated to two groups on the basis of uniformity of established engorged tick burdens. Six calves were treated with doramectin and six received saline solution. From Day -3 to Day 21 post-treatment, individual collections of detached engorged female ticks were made from each calf. In the persistent efficacy study, 12 calves were allocated to two groups of six animals. Six calves were treated with doramectin and six received saline solution. From Day 1 to Day 17 post-treatment, each animal was infested three times a week along the dorsal line with 2500 recently hatched Boophilus microplus larvae, for a total of nine times. From Day 18 to Day 42 post-treatment, daily collections of detached engorged female ticks were made from individual animals. In the therapeutic study, efficacy (reduction of collected engorged female ticks) progressed from 51% at 24 h post-treatment (p.t.) to at least 99% at 4 days p.t., and reached 100% at 8 days p.t. With the exception of one tick that did not lay eggs, recovered from one animal at 11 days p.t., no more ticks were recovered from doramectin-treated calves for the duration of the experiment. For the first 6 days after treatment, only a few detached engorged ticks were collected from treated animals, and their oviposition and hatchability declined rapidly. In the persistent efficacy study, doramectin treatment was highly efficacious in preventing the establishment of Boophilus microplus populations for 20 days after the first ticks completed their cycle in the non-treated group. The oviposition and hatchability of the few ticks that completed their life cycle in the doramectin group were severely reduced.

Purification and characterization of β-N-acetylhexosaminidase from bovine tick Boophilus microplus (Ixodide) larvae

beta-N-Acetylhexosaminidase (HEX, E.C. 3.2.1.52) from larvae of the ixodid tick Boophilus microplus was purified to capillary zone electrophoresis homogeneity, and characterized. Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-200, p-aminobenzyl-N-acetyl-beta-D-thioglucosamine affinity, and Mono-Q FPLC columns. Purification was about 1600-fold, with a yield of 10%, as determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme presented optimum pH 4.7, and optimum temperature 65 degrees C. The molecular weight of non-denatured enzyme was estimated as 127,000 by gel filtration chromatography, and 60,000 in SDS-PAGE. The tick hexosaminidase presented glycosyl residues, as evidenced by binding to Concanavalin-A. Among several p-nitrophenyl glycosides tested as substrate, HEX was active only on p-nitrophenyl-N-acetylglucosaminide and p-nitrophenyl-N-acetylgalactosaminide. The purified enzyme presented immunogenicity in rabbit, and the correspondent antibodies inhibited about 90% of its original, in vitro activity.

Changing patterns of vitellin-related peptides during development of the cattle tick Boophilus microplus

The major components of protein extracts from the cattle tick Boophilus microplus eggs and larvae of various ages were characterized by molecular sieving chromatography, ion exchange chromatography and SDS-PAGE. The fractions analysed showed a changing chromatographic pattern development. A serum raised against the components of a fraction showing characteristics of vitellin strongly reacted in Western blots with the major peptides of extracts from eggs, larvae, gut and ovary. Comparison of patterns obtained by electrophoresis in non-denaturing PAGE, stained with Coomassie blue or with benzidine/hydrogen peroxide, revealed that the major proteins of these extracts are haemoproteins, possibly in different aggregation states or heterogeneous in composition.

Monoclonal antibodies against Boophilus microplus and their effects on tick reproductive efficiency

Four monoclonal antibodies (mAbs) against extracts of embryo and gut tissue obtained from fully engorged Boophilus microplus were produced. The mAb BrBml reacted with different instars and tissues, the BrBm2 recognized only antigens present in gut extract and the mAbs BrBm3 and BrBm4 recognized vitellin. The effect of inoculation of these mAbs into fully engorged Boophilus microplus females was also evaluated. The mAbs BrBm1 and BrBm2 caused a decrease in oviposition of approximately 50% and 70%, respectively, and the mAbs BrBm3 and BrBm4 did not affect reproductive efficiency. This assay may be useful as a low-cost test to provide preliminary information on the possible effects of anti-tick antibodies in damaging ticks before attempting cattle vaccination experiments.

A putative RNA virus in Babesia bovis

Babesia bovis is an intraerythrocytic protozoan that causes bovine babesiosis. Agarose gel electrophoresis of nucleic acids extracted from two isolates of B. bovis reveals, besides bulk DNA, an ethidium bromide-stainable band at about 5.5 kb. Further characterization of the latter with DNase I, RNase and mung bean nuclease suggested it to be a double-stranded RNA. Sonicated parasites were fractionated in a CsCl buoyant density gradient. A sample containing the 5.5-kb RNA was analysed under an electron microscope and a virus-like particle was observed.

Extrachromosomal nucleic acids in bovine babesia

Two kinds of small extrachromosomal nucleic acid elements were found in the bovine babesias, Babesia bovis and B. bigemina. One element with an apparent size of 5.5 kilobase pairs (kbp) is a double stranded RNA related to virus like particles. Another molecule is a double stranded DNA with a molecular size of about 6.2 kbp. Southern blot comparison of restriction DNA fragments of the latter molecule, which is present in both B. bovis and B. bigemina is described.