Determination of disulfide bond pairs and stability in recombinant tick anticoagulant peptide.

Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D.E., Arcuri, K.E., and Vlasuk, G.P. (1990) Science 248, 593-596). The 60-amino acid sequence of TAP shows limited homology to Kunitz-type inhibitors, including cysteines at positions 5, 15, 33, 39, 55, and 59. For detailed biochemical and pharmacological studies, a recombinant version of TAP (rTAP) has been produced in yeast. To determine the arrangement of the disulfide bonds, rTAP was cleaved with trypsin and chymotrypsin and the purified peptides sequenced using a gas-phase sequenator. The positions of the disulfide bonds were assigned by identifying the cycle(s) at which di-phenylthiohydan-toin-cystine was released. The specific disulfide bridges, Cys-5 to Cys-59, Cys-15 to Cys-39, and Cys-33 to Cys-55, are analogous to those in the prototype Kunitz-type inhibitor, bovine pancreatic trypsin inhibitor (BPTI). While treatment of BPTI with dithiothreitol rapidly and specifically reduced one disulfide bond, the reduction of disulfide bonds in rTAP proceeded at a slower rate and appeared to be nonspecific, reaching a maximum of two disulfides reduced. Reduced rTAP derivatized with either iodoacetic acid or iodoacetamide lost 59% of its inhibitory activity. In contrast, BPTI alkylated with iodoacetic acid inhibited trypsin half as well as the iodoacetamide derivative. Although the arrangement of disulfides in the two inhibitors is the same, their susceptibility to reduction is markedly different.

Purification and characterization of an anticoagulant from the salivary glands of the ixodid tick Rhipicephalus appendiculatus.

An anticoagulant isolated from salivary gland extracts of the ixodid tick Rhipicephalus appendiculatus was purified by gel filtration on Sephadex G-100, ion exchange on DEAE-cellulose, aprotinin-Sepharose, and by high-pressure-liquid size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the anticoagulant activity was associated with a protein of an apparent Mr of 65 kDa. The purified molecule had a pI in the range of 8.0-8.5 on chromatofocusing and was stable over a wide pH range, but was heat labile and susceptible to inactivation by trypsin and reductive alkylation. The anticoagulant did not inhibit thrombin-initiated fibrin formation and had no detectable fibrino(geno)lytic or phospholipase-like activities. Although it inhibited factor Xa-induced clotting of bovine plasma, it did not affect the amidase activity of factor Xa toward a synthetic substrate, suggesting that the anticoagulant acts at a site distinct from the active site of factor Xa or on other components of the prothrombinase complex.

[Natural foci of tick-borne encephalitis on the southwestern coast of Sakhalin]. / Prirodnye ochagi kleshchevogo éntsefalita na iugo-zapadnom poberezh'e Sakahlina.

Nucleic acids of Ixodes persulcatus were studied by molecular hybridization in the natural focus of tick-borne encephalitis in Kholmsk District of Sakhalin Province. The studies have shown wide dissemination of viral RNA in the focus. The infectivity of ticks in various sites of habitation varied from 3.5 to 18.5%, their number fluctuating from 0.4 to 300 and more imago per flag-hour. The most active part of the natural focus has been determined using zoological-viral indexes. The viral strain of tick-borne encephalitis has been isolated.

Lectin binding analysis of Argas polonicus tissue glycoproteins.

Proteins of the Malpighian tubules (MT), midgut tissue (MG), salivary glands (SG), internal reproductive organs (RO), epidermis (EP), cerebral ganglion (CG), rectal ampulla (RA) and larval homogenate (LA) of Argas (Argas) polonicus were studied for their antigenicity and lecin affinity using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, lectin affinoblotting and enzyme-linked lectin sorbentassay (ELLSA) techniques. A glycoprotein of 305 kDA was found in all tissues studied. All low molecular weight antigenic proteins recognized by anti-larval immune pigeon serum, except for one of 35 kDA, i.e. the 19-, 21-, 23-, 27-, 34-, and 46- kDa proteins, were shown to be glycoproteins. The glycosylation was shown to be N-linked in all of these antigens, but O-type glycosylation was also demonstrated in the 34-kDa glycoprotein. The correlation between the glycosylation and antigenicity of these proteins is also discussed.

Clinical illness associated with a commercial tick and flea product in dogs and cats.

A commercial flea and tick product containing 9.0% fenvalerate for use in dogs and cats was suspected of causing illness. An acute toxicity study was performed in 10 dogs and 10 cats exposed to the product orally (po) and dermally at differing doses. Samples were obtained for DEET and fenvalerate analysis. Oral dosing of dogs and cats produced severe clinical illness at doses as low as 0.66% of a can (7 ounce spray can)/kg body weight. Dermal application of the product resulted in minor clinical abnormalities in dogs. Oral exposure at 0.5% can/kg body weight resulted in severe illness, and dermal application caused severe illness or death in cats at 20% and 40% of a can/kg body weight. The cats receiving 10% of a can/kg body weight dermally became depressed for several hours but recovered uneventfully. Serum DEET concentrations closely paralleled the clinical signs observed in the animals. Serum concentrations of DEET above 20 ppm were considered diagnostic for intoxication. Urine concentrations of DEET above 1 ppm and tissue (liver, bile, and kidney) concentrations of DEET above 10 ppm were supportive of poisoning; values near 100 ppm were diagnostic for fatal poisoning.

The detection and isolation of a paralysis toxin present in Argas (Persicargas) walkerae.

One-day-old leghorn chickens were used in a laboratory assay to determine the toxicity of crude extracts of the tick Argas (Persicargas) walkerae and of fractions obtained during the isolation procedure. Extracts of unfed and engorged larvae, nymphae and females were tested using this in vivo test system. Only extracts of replete A. (P.) walkerae larvae produced paralysis. A toxic fraction was isolated from replete larval extracts by gel-permeation and ion-exchange chromatography. This fraction with a pI of 4,5, showed 2 major bands corresponding to a Mr of 32 kDa and 60 kDa after SDS-polyacrylamide gel electrophoresis.

Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa.

A low molecular weight serine protease inhibitor (TAP) was purified from extracts of the soft tick, Ornithodoros moubata. The peptide is a slow, tight-binding inhibitor, specific for factor Xa (Ki = 0.588 +/- 0.054 nM). The inhibitor also acts as an anticoagulant in several human plasma clotting assays in vitro. Its amino acid sequence (60 residues) has limited homology to the Kunitz-type inhibitors. However, unlike other inhibitors of this class, TAP inhibits only factor Xa. It had no effect at a 300-fold molar excess on factor VIIa, kallikrein, trypsin, chymotrypsin, thrombin, urokinase, plasmin, tissue plasminogen activator, elastase, or Staphylococcus aureus V8 protease. TAP's specificity and size suggest that it may have therapeutic value as an anticoagulant.

Evaluation of partially purified salivary gland allergens from the Australian paralysis tick Ixodes holocyclus in diagnosis of allergy by RIA and skin prick test.

Two diagnostic assays that detect IgE specific to I. holocyclus were developed using purified sources of allergens. Salivary gland extract was superior to whole body extract in both the radioimmunoassay and the skin prick test. Of the partially purified salivary gland allergens, the 28-KD protein fraction gave the most promising results.

Hybridization model for Rhipicephalus appendiculatus and R. zambeziensis by glucose-P-isomerase isoenzymes.

Hybrids between Rhipicephalus appendiculatus and R. zambeziensis were reared and glucose-phosphate-isomerase isoenzymes were resolved by agarose electrophoresis. By phenotyping hybrids in F1 and F2 generations autosomal transmission of 2 GPI genes was demonstrated. Identification of a hybrid phenotype provides a method for identifying hybrids in field collections.

Mating and behavioural diapause in Ixodes ricinus L.

This study established that in an open rough-grazing habitat in Ireland a large proportion of female ticks mated in the vegetation rather than on the host, confirming observations made by other workers in scrub and forest habitats in Switzerland and Russia. It was found that many ticks active from October onwards had already mated even though they were moulted and apparently derived from the spring population. This activity was at odds with the view that such ticks are prevented from becoming active in autumn by a behavioural diapause. Laboratory and plot experiments involving observations on questing activity and feeding showed that the behavioural diapause of females was abolished by exposure to males. Male ticks did not show a behavioural diapause. In areas of high tick density this mechanism could result in the transfer of ticks from spring to autumn populations and, since the mortality rate of the autumn population is higher, could also contribute to density-dependent population regulation.

Isolation of a neurotoxin from the salivary glands of female Rhipicephalus evertsi evertsi.

A quantitative study of the changes in the protein pattern of the salivary glands of female Rhipicephalus evertsi evertsi during the entire repletion process was undertaken. These results, in conjunction with the previously determined toxic phase, indicated the presence of a toxic protein. The development of a sensitive in vitro assay using a Xenopus nerve-muscle preparation, made it possible to identify toxic phases during feeding and to assay fractions of salivary gland extracts during toxin isolation. Sufficient amounts of electrophoretically and chromatographically homogeneous toxin could be obtained through the use of chromatofocusing, enabling its characterization with respect to molecular weight (68 kDa; determined by gel permeation chromatography), pI (6.00), and amino acid composition. The toxin was inactivated by pronase digestion as well as by antiserum.

Identification of ecdysone 22-long-chain fatty acyl esters in newly laid eggs of the cattle tick Boophilus microplus.

The five major apolar ecdysone esters present in newly laid eggs of the cattle tick Boophilus microplus have been purified by h.p.l.c. The quantities of the apolar esters present in the eggs were increased by administration of ecdysone to the mature females. G.c.-m.s. analysis, as their methyl esters, of the fatty acids released from the apolar ecdysone derivatives by alkali, coupled with positive-ion fast-atom-bombardment m.s. of the intact ecdysone esters, showed that the compounds consisted of a series of fatty acyl esters of ecdysone. The position of esterification of the ecdysone was established by p.m.r. spectroscopy. The combined data show that the novel apolar derivatives of ecdysone consist of the 22-palmitate, -palmitoleate, -stearate, -oleate, and -linoleate esters respectively. Confirmation was obtained by comparison with synthetic ecdysone 22-palmitate. The significance of the ecdysone fatty acyl esters as a possible source of free hormone during embryogenesis is discussed.

Differentiation between three species of Amblyomma ticks (acari: Ixodidae) by analysis of cuticular hydrocarbons.

Cuticular lipids were extracted from three species of Amblyomma ticks in hexane: dichloromethane and analysed by gas-liquid chromatography using both packed and capillary columns. Unlike many of the reported insect hydrocarbons, those of the Amblyomma ticks consisted mostly of branched paraffins and olefins and all three species of ticks are well differentiated on the basis of numbers and carbon-chain lengths of the hydrocarbons. Amblyomma maculatum has more olefins and A. cajennense has more paraffins than the other species, including A. americanum.

Production in ascites fluid of biosynthetically labelled monoclonal antibody to Theileria parva sporozoites.

A hybridoma cell line has been previously produced which secretes monoclonal antibodies able to neutralize sporozoites of Theileria parva, the causative agent of East Coast fever of cattle. Cells from this line were injected intra-peritoneally into pristane-treated BALB/c mice. During the last 4 days of hybridoma cell growth, mice were given 4 daily intraperitoneal injections of a mixture of tritiated amino acids in order to biosynthetically radiolabel the monoclonal antibody being produced in ascites fluid. The specific activity of the antibody obtained was 100 mCi/mmol. The labelled antibody was used to detect, by autoradiography, a surface coat antigen of T. parva sporozoites in cryostat sections of Theileria-infected tick salivary glands. The method allows the preparation of large quantities of biosynthetically radiolabelled immunological probes for the detection of immunoreactive sites in biological specimens.

Immunohistochemical localization of adenosine 3':5'-cyclic monophosphate in female ixodid tick Amblyomma americanum (L.) salivary glands.

Localization of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in alveoli of salivary glands of female Amblyomma americanum (L.) was accomplished with an indirect immunofluorescent technique. Little cyclic AMP fluorescence was seen in Type I alveoli in glands of unfed females but considerable fluorescence was seen in Type I alveoli of glands obtained from females that had fed. The most intense cyclic AMP fluorescence was observed in complex granular cells of Type II and III alveoli in glands of unfed females and glands from females in early stages of tick feeding. In the latter stages of tick feeding an increase in fluorescence in Type III alveoli was observed in cells near the lumen, possibly adluminal interstitial or transformed granular cells.

Improved toxin/antitoxin assays for studies on the Australian paralysis tick Ixodes holocyclus.

Sensitive biological assays of toxin/antitoxin potency have been developed to assist in research on characterization of salivary toxins of the Australian paralysis tick Ixodes holocyclus and on immunity to tick paralysis. The toxin assay utilizes suckling mice (4-5 g); a quantitative paralysis index is applied over a range of doses. The antitoxin assay is based on an in vitro/in vivo neutralization test which required a sensitive toxin assay and methods of standardization of toxin preparations. This assay permits the monitoring of blood antibody levels in animals during the course of development and loss of immunity and is assisting a study into the feasibility of producing an anti-paralysis vaccine. The method also allows standardization of commercial tick paralysis antiserum. The methods and applications are described and comparisons made with previous assays. Sample data are examined statistically by regression and variance analyses; parallelism of dosage-response lines is tested and relative toxicities (toxin) or potencies (antiserum) calculated.