Experimental vaccination of sheep and cattle against tick infestation using recombinant 5'-nucleotidase.

Limited prior evidence suggests that 5'-nucleotidase, an ectoenzyme principally located in the Malpighian tubules of the tick Rhipicephalus (Boophilus) microplus, could be an effective antigen in an anti-tick vaccine. To assess this, recombinant 5'-nucleotidase was expressed in Escherichia coli and used in vaccination trials with both sheep and cattle. Vaccinated sheep were challenged with freshly moulted adult ticks. Those with high titres of anti-nucleotidase antibodies showed significant protection against tick infestation, although protection was less than that found with the previously characterized antigen, Bm86. Cattle were vaccinated, in separate groups, with 5'-nucleotidase, Bm86 and both antigens combined. Cattle, as the natural host, were challenged with larval ticks. Although Bm86 showed typical efficacy, no significant protection was seen in cattle vaccinated with 5'-nucleotidase. Cattle receiving a dual antigen formulation were no better protected than those receiving Bm86 alone. One possible reason for the difference between host species, namely antibody titre, was examined and shown to be an unlikely explanation. This demonstrates a limitation of using a model host like sheep in vaccine studies.

Control of parasitic disease using vaccines: an answer to drug resistance?

Antiparasitic drugs have been used successfully to control parasitic diseases in animals for many years, as they are safe, cheap and effective against a broad spectrum of parasites. One drawback of this success appears to be the emergence of drug resistance in many target parasites. Moreover, issues of residues in the food chain and environment have arisen, which threaten their sustained use. Control methods in which vaccines would have a central role provide attractive alternatives. However, while attenuated parasite vaccines have been successful, sub-unit vaccines are still rare. The advent of new techniques in molecular biology allows the elucidation of entire parasite genomes and the identification of individual genes. It is envisaged that a further understanding of parasite genes and the role of their products in parasite biology may lead to the identification of useful antigens, which could then be produced in recombinant systems. However, for this aim to be realised, continued investment in basic research on the complex interplay between parasite and host will be necessary.

Simultaneous isolation of bovine eosinophils and neutrophils on gradients of Percoll.

Bovine eosinophils and neutrophils have been isolated from venous blood using a two-step procedure which involves initial preparation of a granulocyte-rich fraction by centrifugation and osmotic shock followed by sensity gradient centrifugation on Percoll. Good yields of both cell types are obtained with purities of 90% or better. The isolated cells are viable and actively phagocytic. The effects of phagocytosis on the metabolism of carbon-1 of d-glucose and the incorporation of iodine have been measured and several differences between bovine cells and those of other species were noted.

Protein phosphorylation by intact Babesia bovis.

Babesia bovis rapidly incorporated [32P]orthophosphate into more than 30 polypeptide chains, the majority of which appeared to be membrane or particle bound. Phosphorylation occurred more rapidly in free parasites than in parasitized erythrocytes, suggesting the acceptor polypeptides were either babesial in origin or erythrocyte proteins were intimately associated with the parasite. Some characteristics of the phosphorylation system are described. The phosphorylation was little affected by dibutyryl cAMP or dibutyryl cGMP but was strongly inhibited by the calcium ionophore A23187, which also inhibited protein synthesis. Two dimensional electrophoresis of B. bovis extracts after pulse labelling with [35S]methionine or [32P]orthophosphate showed that all polypeptides phosphorylated in a 30 min period were also at least partially synthesized de novo within that period, suggesting that the phosphorylated species were babesial in origin.

Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana.

Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.

Peritrophic matrix proteins.

The peritrophic matrix (or peritrophic membrane) lines the gut of most insects at one or more stages of the life cycle. It has important roles in the facilitation of the digestive processes in the gut and the protection of the insect from invasion by microorganisms and parasites. The traditional view of the peritrophic matrix as a relatively insert sieve, composed largely of proteins and glycosaminoglycans embedded in a chitinous matrix, is under revision as more is learned about the molecular characteristics of the peritrophic matrix proteins. This review summarizes emerging knowledge of the main protein constituents of the peritrophic matrix. The availability of the first sequences of integral peritrophic matrix proteins has coincided with the explosion of information in sequence databases. It is therefore possible to examine common structural themes in this family of proteins as well as in proteins of unknown location and function from a variety of other insects, nematodes and viruses. The review concludes with speculation about the biological functions of the proteins in this matrix.

Endocytosis by digest cells of the cattle tick Boophilus microplus: regulation by protein kinase C.

Endocytosis of fluorescently-labeled bovine serum albumin by digest cells of the gut of the cattle tick Boophilus microplus is inhibited by approx 60% in the presence of the tumour promoter 12-O-tetradecanoylphorbol 13-acetate. The results are consistent with a role for protein kinase C in regulating the uptake of blood meal by digest cells. Protein kinase C activity has been measured in the digest cell and the amount of enzyme has also been determined using a phorbol ester binding assay. The presence of a small number of specific protein kinase C substrates in the plasma membrane of the digest cell has been demonstrated. Preliminary experiments indicate that one of these substrates, a protein of approximately 30 kDa, is an integral membrane protein, part of which is exposed on the extracellular surface of the digest cell.

The nucleotidase of Boophilus microplus and its relationship to enzymes from the rat and Escherichia coli.

Boophilus microplus contains a nucleotidase-like enzyme which is able to hydrolyze a range of nucleoside 5'-mono-, di- and triphosphates to the nucleoside. Its relationship to several other nucleotide hydrolyzing enzymes has been explored. Limited peptide sequencing shows similarities to both mammalian nucleotidases and the Escherichia coli uridine diphosphate sugar hydrolase. The tick enzyme also hydrolyzes UDP-glucose, though by a mechanism different to that of the bacterial enzyme. On the other hand, it resembles the mammalian nucleotidases in that there is evidence that it is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor.

Identification and molecular characterisation of a peritrophin gene, peritrophin-48, from the myiasis fly Chrysomya bezziana.

The peritrophic matrix lines the midgut of most insects and has important roles in digestion, protection of the midgut from mechanical damage and invasion by micro-organisms. Although a few intrinsic peritrophic matrix proteins have been characterised, no direct homologues of any of these proteins have been found in other insect species, even closely related species, suggesting that the peritrophic matrix proteins show considerable sequence divergence. We now report the identification of the cDNA and genomic DNA sequences of a Chrysomya bezziana homologue of the Lucilia cuprina intrinsic peritrophic matrix protein, peritrophin-48. The gene for C. bezziana peritrophin-48 spans 1315 bp and consists of three exons (65, 560 and 690 bp, respectively) separated by introns of 566 and 72 bp. The transcriptional start site, identified by a consensus of cDNA clones and primer extension analysis, is probably located 58 bp upstream from the start codon. However, there may be multiple start sites for transcription. Two potential TATA boxes and a consensus arthropod transcription initiator are located within 134 bp of sequence upstream of the putative transcriptional start site suggesting that this region contains the gene promoter. Immuno-fluorescence localization demonstrated that C. bezziana peritrophin-48 was localised to the larval peritrophic matrix. Protein fold recognition analysis indicated structural similarities between peritrophin-48 and wheatgerm lectin. As wheatgerm lectin binds chitin, this result suggested that C. bezziana peritrophin-48 may also bind chitin, a constituent of the peritrophic matrix. Chitin binding studies with a recombinant peritrophin-48 protein confirmed that it binds chitin. A Drosophila melanogaster homologue of peritrophin-48 encoded in an EST and a genomic sequence was also identified. The pairwise percentage identities of the deduced amino acid sequences for the peritrophin-48 homologues from the three higher Dipteran species were relatively low, ranging between 32 and 42%. Despite this sequence variability, the predicted structure of these proteins, dictated by five domains, each containing a characteristic distribution of six cysteines, was strictly conserved. It is concluded that considerable sequence variation can be tolerated in this protein because of the constraints imposed on the structure of the protein by an extensive disulphide bonded framework.

Carboxydipeptidase from Boophilus microplus: a "concealed" antigen with similarity to angiotensin-converting enzyme.

A protein, Bm91, which was first identified as a protective vaccine antigen from the tick Boophilus microplus, has regions of very strong amino acid sequence similarity to mammalian carboxydipeptidases or angiotensin converting enzymes (ACE; E.C. 3.4.15.1). This protein is now shown to share many biochemical and enzymatic properties with mammalian carboxydipeptidases. It is enzymatically active in a conventional assay for ACE using hippuryl-Gly-Gly as substrate. The hydrolysis of the C-terminal nonapeptide of the insulin B chain proceeds by sequential removal of carboxy-terminal dipeptides. The similarities extend to the dependence of activity on pH and added salt. Bm91 is inhibited by two well-characterized inhibitors of the mammalian enzymes, the drug Captopril and a nonapeptide, and the inhibition occurs in similar concentration ranges to those effective with the mammalian enzymes. However, the natural substrates of the tick enzyme are unknown. Angiotensin I itself is a poor substrate and the enzyme's natural substrates are likely to be one or more of the pharmacologically active peptides occurring in insects and arthropods.

Biochemical and molecular characterization of serine proteases from larvae of Chrysomya bezziana, the Old World Screwworm fly.

The diversity of serine proteases secreted from Chrysomya bezziana larvae was investigated biochemically and by PCR and sequence analysis. Cation-exchange chromatography of purified larval serine proteases resolved four trypsin-like activities and three chymotrypsin-like activities as discerned by kinetic studies with benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Amino-terminal sequencing of the three most abundant fractions gave two sequences, which were homologous to other Dipteran trypsins and chymotrypsins. Analysis of products generated by PCR of cDNA from whole larvae using specific primers based on the amino-terminal sequences and generic serine protease primers identified 22 different sequences, while phylogenetic analysis of the deduced amino acid sequences differentiated two trypsin-like and four chymotrypsin-like families. Phylogenetic comparisons with Dipteran and mammalian serine protease sequences showed that all the Chrysomya bezziana sequences clustered with Dipteran sequences. The Chrysomya bezziana chymotrypsin-like sequences segregated within a Dipteran cluster of chymotrypsin sequences, but were well dispersed amongst these sequences. The largest Chrysomya bezziana serine protease family, the trypB family, clustered tightly as a group, and was closely related to a Lucilia cuprina trypsin but distinct from Drosophila melanogaster alpha and beta trypsins. The trypB family contains ten highly homologous sequences and probably represents an example of concerted evolution of a trypsin gene in Chrysomya bezziana.

Assay of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum.

It has been proposed that Clostridium aminobutyricum contains an enzyme catalyzing an unusual reaction: the dehydration of 4-hydroxybutyryl-CoA to vinylacetyl-CoA. 4-Hydroxy-[3-3H]butyric acid has been prepared which allows the activity of this enzyme to be assayed in the presence of acetyl-CoA under anaerobic conditions by the release of tritiated water. Initial characterization of the enzyme from C. aminobutyricum has shown it to be largely membrane or particle bound in the crude lysates. It can be solubilized in detergent. It is inactivated by oxygen, but stable under anaerobic conditions. Only 49 +/- 2% of the label is removed after enzyme-catalyzed equilibration with water. This stereospecific release is consistent with the formation of vinylacetyl-CoA and excludes a vitamin B12 coenzyme-dependent rearrangement to 3-hydroxybutyryl-CoA followed by dehydration to crotonyl-CoA.

Novel vaccination for control of the Babesia vector, Boophilus microplus.

An antigen was isolated from the tick gut wall. Antibody to this antigen bound to the tick gut cells when the ticks fed on immunized animals. Gut function in the ticks and tick development were inhibited.

Bm86 antigen induces a protective immune response against Boophilus microplus following DNA and protein vaccination in sheep.

Vaccination of sheep with a plasmid bearing the full length gene for the tick antigen Bm86 either alone or co-administered with plasmid carrying the ovine genes for the cytokines, granulocyte and macrophage colony stimulating factor (GM-CSF) or interleukin (IL)-1beta induced a relatively low level of protection against subsequent tick infestation. This tick damage reached statistical significance only for the groups which were vaccinated with plasmid encoding for Bm86, co-administered with plasmid encoding for ovine GM-CSF. Antibody titres measured against Bm86 were also low in all groups injected with the Bm86 DNA vaccine. Antibody production and anti-tick effect were significantly less than that achieved by two vaccinations with recombinant Bm86 protein. In all cases only a low level of antigen-specific stimulation of peripheral blood lymphocytes was recorded, as measured either by the incorporation of tritiated thymidine or the release of IFN-gamma. Injection of DNA encoding for Bm86, either alone or with co-administered cytokine genes, did however prime for a strong subsequent antibody response following a single injection of recombinant Bm86 protein in adjuvant. Antibody production nevertheless appeared to be slightly less effective than following two vaccinations with recombinant protein. The persistence of antibody following vaccination was the same regardless of the method of primary sensitization. In all cases the half-life of the antibody response was approximately 40-50 days indicating that, in contrast to results reported in mice, DNA vaccination in sheep did not result in sustained antibody production.

Novel vaccines for ectoparasites.

Novel vaccines against ectoparasites have the potential to be cost-effective new technology for pest control that avoids some of the real and perceived problems with insecticide and acaricide usage. Nevertheless, their development is in its infancy. A vaccine against the cattle tick Boophilus microplus, the world's first vaccine against an ectoparasite, is in field use in Australia. Considerable effort had gone into the development of a vaccine against the sheep blowfly Lucilia cuprina, while other vaccines are at an earlier stage of development. The identification of critical antigens and their production as effective recombinant proteins remains the greatest hurdle. Characteristics of the few known antigens and the mode of action of the protective immune response are discussed. Development of further vaccines will depend on recognition of likely antigenic targets. The efficacy of such vaccines will depend on the characteristics of the target species, in particular its digestive biology and the way in which the novel vaccine impacts on the parasite population.

The molecular revolution in the development of vaccines against ectoparasites.

Over the last decade, the application of a spectrum of molecular techniques has begun to revolutionise our understanding of protective immune responses to ectoparasites and the targets for those responses. The catalogue of potential and actual protective antigens characterised in detail is slowly expanding. The validity of regarding such antigens as generic and capable of cross-species protection is being explored. The immune interactions between host and parasite are being studied at a molecular rather than cellular level. All this should contribute to the eventual development of a range of recombinant vaccines, though important scientific limitations remain. These range from the innate susceptibility of individual parasite species to immunological attack, which can only be assessed on a case by case basis, to our ability to produce the desired recombinant antigens and to elicit and maintain the necessary immunological responses.

Prostaglandin E2 production by the cattle tick (Boophilus microplus) into feeding sites and its effect on the response of bovine mononuclear cells to mitogen.

Prostaglandin E2 (PGE2) secretion by the cattle tick Boophilus microplus into feeding sites was quantified. It was detected by the in vitro tube feeding experiment and it was determined that a semi-engorged female tick could produce and transmit 1.8 ng PGE2 into the feeding site. Using the in vitro membrane feeding system, newly molted adult ticks were also shown to secrete 0.04-0.15 ng PGE2 into the feeding site; however, female ticks produced more PGE2 than male ticks. The immune suppressive effect of PGE2 in the saliva of B. microplus on the bovine mononuclear cells (MNC) was also examined. PGE2 in the saliva was suspected of being a major component that inhibited the blastogenic response of MNC to a T-cell mitogen phytohemagglutinin. As bovine MNC are sensitive to low level concentration of PGE2, the PGE2 transmitted into feeding sites was suspected to be sufficient to produce physiological effects on the bovine host.

Effects of cattle tick (Boophilus microplus) infestation on the bovine immune system.

The immunosuppressive effect of experimental Boophilus microplus infestation on bovine peripheral blood lymphocytes (PBL) and on host antibody production to a protein antigen (ovalbumin) was examined. Boophilus microplus infestation caused a marginal decrease in the percentage of T lymphocytes in PBL, which was observed in both lightly (5000 larvae) and heavily (40,000 larvae) infested cattle, and began at the second infestation and continued until the end of the fourth infestation. The percentage of B lymphocytes in heavily tick-infested cattle was less than that in non-infested control cattle after the fourth infestation. The response of PBL from tick-infested cattle to phytohemagglutinin (PHA) was always less than that of tick-free cattle after the second infestation. No noteworthy differences were detected between the three stages of tick infestation, that is, 1 week before the peak of adult engorgement, the middle of the peak and 1 week after all ticks had dropped. Boophilus microplus saliva (100 microliters ml-1) suppressed 47% of the response of bovine PBL to PHA in vitro. This suppressive effect of saliva may contribute to the lower responsiveness of PBL from tick-infested cattle. Antibody production by tick-infested cattle was examined during the third and fourth heavy tick infestation. Tick-infested cattle showed a diminished response against ovalbumin after the second immunization. The immunosuppressive effects of tick infestation may play an important role in tick survival or in the transmission of tick-borne diseases in the field.

Reduced oviposition of Boophilus microplus feeding on sheep vaccinated with vitellin.

The most abundant protein present in Boophilus microplus eggs, vitellin, was isolated and purified as a non-covalent complex of six glyco-polypeptides of Mr 44-107kDa. The protein complex bound haem. Immuno-blots demonstrated that antibodies raised to vitellin recognised a 200kDa polypeptide in the haemolymph of adult female ticks. This is consistent with the general proposal that in arthropods vitellin is derived by proteolytic processing from a large precursor protein, vitellogenin. In parallel with this study, an 80kDa glycoprotein (GP80) was independently purified from larvae of B. microplus using efficacy in vaccination trials as an assay. Antibodies to GP80 also recognised a 200kDa protein in the haemolymph of ticks and a major 87kDa polypeptide present in the vitellin complex. Conversely, antibodies to purified vitellin recognised GP80. The amino-terminal amino acid sequences of the 87kDa vitellin polypeptide and GP80 were identical for at least the first 11 residues and internal peptide sequences from both polypeptides were co-located in a single but incomplete deduced amino sequence of B. microplus vitellogenin. Thus, GP80 is a processed product from vitellogenin and highly related to but not completely identical with the 87kDa vitellin polypeptide. Vaccination trials in the model host sheep were performed with purified vitellin and GP80. Sheep vaccinated with either purified vitellin or GP80 returned significantly reduced numbers of engorged female ticks with decreased weights and reduced oviposition. In contrast, sheep vaccinated with recombinant hexahis-GP80, which was incorrectly folded and not glycosylated showed no significant effects on ticks. It was concluded that vitellin and GP80 could induce immune responses that partially protect sheep from the tick, B. microplus. However, critical protective epitopes are associated with the folding of the protein and/or the oligosaccharides attached to it.