Babesiosis. Diagnostic pitfalls.

A case of babesiosis complicated by quinine-induced hemolysis is described. A splenectomized woman contracted babesiosis after visiting an endemic area. The patient presented with high fevers and minimal hemolysis. While she was on treatment, the hemolysis increased. The increased hemolysis was initially attributed to babesiosis, but after additional evaluation quinine therapy was found to be the cause. In the setting of this brisk hemolysis, there was an increase in red blood cell inclusions. At first, the inclusions were thought to be Babesia, but iron stain demonstrated that many of the inclusions were hemolysis-induced Pappenheimer bodies. The role of the clinical laboratory in sorting out this confusing picture is presented.

Vector competence of the Australian paralysis tick, Ixodes holocyclus, for the Lyme disease spirochete Borrelia burgdorferi.

Clinical and serologic evidence of Lyme disease in Australia, including the typical rash, erythema migrans, has been reported. The vector tick transmitting Borrelia burgdorferi in Australia, however, has not been determined. The Australian paralysis tick, Ixodes holocyclus, is a logical candidate vector of the Lyme disease spirochete in Australia; therefore, we tested the ability of I. holocyclus to acquire and maintain a North American isolate of B. burgdorferi. Larval I. holocyclus ingested spirochetes, but none of 84 derived nymphs were infected. These experiments should be repeated with Australian strains of spirochetes.

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.

An immunoblot technique for identification of allergens of the Australian paralysis tick Ixodes holocyclus.

The blocking conditions appropriate for Western blot identification of allergens of the Australian paralysis tick, Ixodes holocyclus, have been investigated. The results suggest that human serum is a potent blocking agent which may have wide application in the immunodetection of antigens.

Tick/host interactions for Ixodes holocyclus: role, effects, biosynthesis and nature of its toxic and allergenic oral secretions.

The Australian paralysis tick Ixodes holocyclus occurs along the eastern coast of Australia. Its interaction with a wide variety of hosts causes a serious toxicosis (tick paralysis) in domestic pets and livestock (occasionally in wildlife and humans) as well as hypersensitivity reactions in humans. Tick paralysis in animals is usually fatal in the absence of speedy antitoxin treatment and human hypersensitivity may result in life-threatening anaphylaxis. The protection of such hosts against toxic or allergic effects by vaccination or desensitisation respectively has been the objective of most of our recent research. The role, biosynthesis and nature of the paralysing toxin (holocyclotoxin) and of the allergens is gradually being elucidated. In this review, some emphasis has been placed on recent research on the interactions of humans with this tick and on the partial characterisation of the allergens.

Allergic reactions to the Australian paralysis tick, Ixodes holocyclus: diagnostic evaluation by skin test and radioimmunoassay.

Allergic reactions to Ixodes holocyclus are well recognized but poorly defined. Tick-bite reactions in 42 individuals in this study fell into six classes. Skin-prick tests and radioimmunoassay (RIA) indicated that all systemic hypersensitivity (class 3) and atypical reactions (class 4) were IgE-mediated. Some 73% of the large local reactions (class 2) and only 12.5% of the small local reactions (class 1) were associated with IgE specific for tick allergens. Subjects who reported heavy exposure to tick-bite were more likely to have positive RIA values (P less than 0.05). There was an association between the individual's atopic status and tick allergy (P greater than 0.05).

Detection in allergic individuals of IgE specific for the Australian paralysis tick, Ixodes holocyclus.

Three techniques were developed to detect IgE specific to Ixodes holocyclus. The radioimmunoassay (RIA) was found to be superior to both radioallergosorbent test and enzyme immunoassay techniques. Validation of the RIA technique was provided by dilution experiments and by a blind study involving 13 volunteers; the RIA graph was linear over a wide range of concentrations. The RIA ratio clearly discriminated between those volunteers with and without systemic allergic reactions and helped to identify borderline cases showing local or cutaneous allergic reactions. It appears to be suitable for diagnostic and research applications.

Isolation and immunological characterisation of allergens from salivary glands of the Australian paralysis tick Ixodes holocyclus.

Radioimmunoassay and Western blot analysis showed that salivary gland extract (SGE) prepared from Ixodes holocyclus was a more concentrated source of allergens than whole body extract. Isolation and partial immunological characterisation of allergens was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of SGE, electroelution and Western blotting. Immunoblotting using allergic sera showed that partially purified SGE from I. holocyclus contained two common allergens of approximately 28 and 35 kilodaltons and several minor allergens. The two most common allergens gave radioimmunoassay ratios significantly greater than the less common allergens. The immunological purity of electroeluted allergens was confirmed by further SDS-PAGE, silver staining and Western blotting.

Tick-paralysis toxoid: an effective immunizing agent against the toxin of Ixodes holocyclus.

Glutaraldehyde treatment of the toxin of the Australian paralysis tick Ixodes holocyclus produced a potent, immunogenic toxoid. Three subcutaneous injections (2 to 4 weeks apart) into rabbits produced very high antitoxin titres in their serum, although circulating antitoxin titres declined in the absence of stimulation. Twenty weeks later, immunized rabbits were unaffected by lethal doses of toxin injected subcutaneously.

Tick (Ixodes holocyclus) paralysis in the dog--induction of immunity by injection of toxin.

Three beagle dogs were immunised against the paralysing effects of holocyclotoxin, using toxic extracts derived from salivary glands of the Australian paralysis tick, Ixodes holocyclus. Upon subsequent challenge with four ED50's of toxin the animals were completely immune, whereas during the early stages 2 of 3 dogs became paralysed after injection with 0.5 ED50. Three immunised beagles, on which 15 I. holocyclus ticks had fed, showed no symptoms but a control beagle became paralysed after 4 I. holocyclus females fed on it. Treatment was instituted immediately upon the onset of clinical signs and the dog recovered uneventfully.

Immunization of rabbits to produce high serum titres of neutralizing antibodies and immunity to the paralyzing toxin of Ixodes holocyclus.

Rabbits have been immunized against the effects of the paralyzing toxin of the Australian paralysis tick Ixodes holocyclus by injecting them with preparations extracted from tick salivary glands. Immunized rabbits were able to withstand doses of toxin known to kill unimmunized rabbits. Neutralizing antibodies were detectable in serum after 2-4 doses of the crude extract or of the relatively pure antigenic fraction. When injections were continued at intervals of from 2-7 weeks, hyperimmunity was retained for at least 68 weeks. Hyperimmune serum, reaching a very high titre of neutralizing antibodies, was obtained after 3-6 injections. Titres tended to decline when boosting ceased, but after a "rest period' high titres were restored by further boosting with normally lethal doses of toxin. No symptoms of tick paralysis developed despite low titres prior to boosting. Thus, once hyperimmunity had been established, high titres of circulating antibodies were not immediately essential for immunity to tick paralysis. An IgG fraction was obtained from rabbit serum using a Protein A-Sepharose method; 33.4 micrograms of IgG protein fully neutralized, and 19.5 micrograms IgG half neutralized, 1 micrograms of crude toxin protein. This procedure with rabbits may permit the production of a purified tick-paralysis antitoxin more suitable for human use than the existing antitoxin based on canine hyperimmune serum.

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.

Inheritance of resistance to chlorpyrifos in the Mt Alford strain and to diazinon in the Gracemere strain of the cattle tick (Boophilus microplus).

Reciprocal crossing of the Mt Alford (A) strain of the cattle tick B. microplus with a susceptible (S) strain and phenotype analysis of F1, testcross and F2 progeny showed that high chlorpyrifos resistance in strain A was due to two genes that were complementary and jointly exhibited incomplete dominance. Diazinon resistance in the Gracemere (G) strain appeared to be similarly inherited. The 'average' degree of dominance ('average dominance', Dav) of high chlorpyrifos resistance over susceptibility, exhibited by F1 hybrids from A X S reciprocal crossings, was +0.54 on a -1 to +1 scale and was not significantly different from the parametric value of +0.5 (semi-dominance). The corresponding Dav values revealed by G X S crossings were +0.42 for diazinon resistance (significantly less than +0.5) and -0.031 for chlorpyrifos resistance (not significantly different from zero and therefore exhibiting zero dominance/recessivity). Resistance factors for chlorpyrifos in strains A and G for homozygotes were 74 and 35, respectively, and for F1 hybrids were 25-29 and 5-7, respectively. The resistance factors for diazinon in strain G for homozygotes and F1 hybrids were 174 and 37-41, respectively.

Norepinephrine as principal catecholamine in a specific neurone of an invertebrate (Boophilus microplus: Acarina).

Norepinephrine has been identified as the principal catecholamine in individual neurons of the synganglion of an arthropod, the cattle tick Boophilus microplus. This suggests that norepinephrine may have a hitherto unsuspected major physiological role in at least one group of invertebrates.