The Australian paralysis tick, Ixodes holocyclus causes a potentially fatal paralysis in domestic animals, livestock and humans with companion animals (mainly dogs) most commonly affected. Current treatment regimes include administration of a commercial tick anti-serum (TAS), prepared as hyperimmune serum in dogs, to neutralise the effects of the toxin. However, each new batch must be standardised using an expensive and highly subjective bioassay performed in neonatal mice. There is currently an urgent need for a more cost effective and rapid in vitro assay that can be more objectively and accurately quantified. Further understanding of the composition of the toxin molecule is also required to develop toxin-specific reagents necessary for these assays. One of the main objectives of this study was to develop a suitable immunoassay to replace the existing mouse bioassay for assessing batches of tick anti-sera for use in tick paralysis therapy in dogs. Initially an enzyme-linked immunosorbent assay (ELISA) was established to detect and quantify antibody specific for I. holocyclus toxin in dog sera. Using a partially purified antigen extracted from I. holocyclus salivary glands, good discrimination was achieved between reactive (hyperimmune) and non-reactive (naïve) sera. The hyperimmune dog sera reacted very strongly with the antigen compared to negligible reactions of serum from dogs not exposed to I. holocyclus. The reactions of hyperimmune sera were also significantly weaker to a non-toxin antigen control extracted from the salivary glands of the non-toxic tick Rhipicephalus microplus, indicating the assay was detecting toxin-specific responses. Furthermore, each of the hyperimmune sera that reacted strongly and specifically with the I. holocyclus antigen in the ELISA also strongly neutralised toxin in the mouse bioassay. Together these findings support the suitability of this ELISA for assessing the potency of batches of commercial dog hyperimmune sera for use as therapy for tick paralysis in dogs. Sera from dogs that were experimentally infested with ticks and sera from patient dogs, presenting at veterinary clinics with signs of tick paralysis, were also screened for antibodies to I. holocyclus antigen using the ELISA. Twenty-eight out of 29 sera from animals with single or multiple exposures to ticks failed to recognise the I. holocyclus antigen indicating the ELISA is not suitable as a diagnostic test to detect toxin-specific antibodies in animals with limited exposure to I. holocyclus infestation. A panel of toxin-specific monoclonal antibodies (mAbs) was produced as research tools to analyse and purify tick toxin components. Rats were successfully immunised against tick toxin using a combination of inoculation of partially purified salivary gland antigen and exposure to tick infestation. The latter approach preserved the native confirmation of the toxin using a natural route of immunisation and rats were chosen due to their high tolerance of multiple tick infestations over several days. While fusion of rat spleen cells with mouse myeloma cells has been reported several times in the literature, the resulting hybridomas are unstable with fastidious culture requirements. Optimisation of the culture conditions revealed that most rat-mouse hybridoma lines grew best in serum-free medium supplemented with 5% foetal bovine serum. Of 600 hybridomas produced, only 12 were shown to be specific for the Ixodes antigen, as determined by ELISA. A selection of these hybridomas representing various patterns of affinity and/or antigen specificity were further analysed for toxin-neutralising ability in a mouse bioassay. Notably, the most potent toxin-neutralising mAb in mice, showed a specific but relatively moderate reaction to Ixodes antigen in the ELISA. The most potent toxin-neutralising mAbs inactivated toxin as strongly as the commercial TAS used for immunotherapy in dogs with tick paralysis. This suggests that mAbs may present an alternative source of immunotherapy, providing a potentially endless supply of a highly consistent reagent and negating the need to use live animals for both the production of tick antiserum and the continual testing of reagent batches. The toxin-neutralising mAbs were also used to analyse I. holocyclus toxin in polyacrylamide gel electrophoresis (PAGE) and Western blot to identify specific toxin proteins. The most potent neutralising mAbs consistently recognised high MW proteins (100-200 kDa) in a smeared pattern. Although this was contrary to previous reports of low molecular weight components (3-5 kDa) in holocyclotoxin, this study was the first to use mAbs prepared to native toxin. The large molecular weight structures likely represent presucursors to, or complexes of the smaller peptides, previously identified. When the Toxin-neutralising mAbs were assessed as ligands to affinity purify toxin components from crude Ixodes SG extracts, toxin components of 110 and 32 kDa were consistently identified. These purified proteins represent good candidates for N-terminal sequencing to further identify the toxin components in I.holocyclus salivary glands.

Thesis (M.S.)--Michigan State University. Dept. of Fisheries and Wildlife, 2008. / Title from PDF t.p. (viewed on July 30, 2009) Includes bibliographical references (p. 176-183). Also issued in print.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 284-323.

Two murine models of acute inflammation, namely thioglycollate-induced peritonitis and carrageenan-induced paw edema, were optimized using non-steroidal anti-inflammatory drug indomethacin and corticosteroid dexamethasone. During the optimization phase, the presence of neutrophils, monocytes, macrophages, eosinophils, B cells and T cells in the peritoneal cavity at various time points after injection of thioglycollate medium was assessed via multicolor flow cytometry. Moreover, two different thioglycollate media (suppliers BD and Sigma-Aldrich) were compared for their ability to induce an inflammatory response. The optimization of thioglycollate-induced peritonitis and carrageenan-induced paw edema was followed by the evaluation of the anti-inflammatory activity of Ixodes ricinus cystatins G1 and G9 in both mouse models.

The aim of the study was to analyze the infectious potential of the newly described species, B. americana and B. carolinensis, studied on the laboratory model mammals mice. Our goal was to analyze and compare the vectorial capacity of two different tick vectors, Amblyomma americanum and Ixodes ricinus, in acquiring and transmition of both spirochete species to the host. The results of this study confirmed that ticks A. americanum and I. ricinus are capable to maintain and transmit B. americana and B.carolinensis.We confirmed that both analysed spirochete species, B. carolinensis and B. americana, showed the potential to develop the disease in laboratory model mammal, which indirectly support the fact that both spirochete species might be concidered as the risk factors in the area where they are distributed. Our results shows that A. americanum is able to transmit both spirochete species, which increases that risk of acquiring the Lyme disease to human population in the area of distribution of A. americanum

Borrelia afzelii differential gene expression in the course of tick blood-feeding, and during chronic infection in mice was studied. Temperature effect on B. afzelii gene expression and infectivity was investigated. Infection rates of mice immunized with B. afzelii tick gut antigen at various stages of tick blood-intake were analyzed. This work was funded by the Grant Agency of the Czech Republic, Project No. 17-27393S to Radek Šíma.

This thesis was focused on the identification and characterization of the salivary proteins from Ixodes ricinus, the European vector of Lyme disease and tick-borne encephalitis causative agents. In the first part of this work, the the transcriptomic approach was used in order to identify and describe I. ricinus salivary proteins. The second part is dealing with functional and structural characterization of the salivary protein named IRS-2 (I. ricinus serpin-2).

The Lyme disease causing spirochaete Borrelia burgdorferi sensu lato is transmitted by ticks within the genus Ixodes. These ticks are liberal host seekers and parasitise mammals, birds and reptiles. Prior to this study, the distribution of I. ricinus ticks and Lyme disease was thought to be restricted to the southern half of Sweden. On the island Norrbyskär, located in the Bothnian Gulf, there were reports of a high incidence of tick infestation on humans. To investigate the occurrence of B. burgdorferi s.l. in these ticks and to characterise presumptive isolates at the molecular level we sampled a number of I. ricinus ticks. Three different isolates were obtained from two different ticks, NBS16 from a nymph and NBS23a and NBS23b from an adult female tick. The seabird associated tick I. uriae is circumpolar distributed in both hemispheres. On the island Bonden, which house one of the largest seabird colonies in the Baltic Sea, I. uriae were collected and surveyed for spirochaetes. One isolate of B. burgdorferi s.l. was obtained. This B. burgdorferi s.l. isolate is identical to the Lyme disease Borrelia strain NBS16 isolated from Norrbyskär. To investigate the role of seabirds in the epidemiology of B. burgdorferi s.l., I. uriae were collected from seabird colonies in the southern and northern hemispheres. Borrelia DNA was extracted from the ticks and from cultured spirochaetes. Sequence analysis of the flagellin gene revealed that the DNA obtained was from B. garinii, regardless of the geographical origin of the sample. Identical fla gene fragments in ticks collected in both hemispheres indicate a transhemispheric exchange of B. garinii. A marine ecological niche and epidemiological route for Lyme disease Borrelia are proposed. The prevalence of B. burgdorferi s.l. infected ticks on migrating passerine birds was studied. A total of 22, 998 birds were caught and examined for ticks. The presence of spirochaetes in the 967 collected ticks was determined by DNA amplification by PCR on all ticks. To determine which B. burgdorferi s.l. species were present, classification was performed by DNA amplification using species-specific 16S rDNA primers and by DNA sequencing. Flagellin gene sequences of all species of B. burgdorferi s.l. previously recorded in Europe were found. B. garinii was the most prevalent. These data support the notion that passerine birds are at least partly responsible for the distribution of Lyme disease Borrelia spirochaetes in Europe. To elucidate the distribution of B. burgdorferi s.l. in subarctic regions, strains isolated from I. ricinus and I. uriae ticks found on islands in the northern Atlantic and Baltic Sea were characterised molecularly. All isolates were verified as B. garinii by 16S-rRNA gene analysis and immunoblotting using monoclonal antibodies specific for the outer surface proteins A and C. Three ribotypes (RT's) of B. garinii were found. The I. ricinus associated RT1 is phenotypically the most heterogeneous. RT2 is restricted to the islands in the northern Baltic Sea, whereas RT3 was also recovered from ticks found on islands in the North Atlantic. The heterogeneity of the B. garinii population in the Baltic Sea might be influenced by two geographically opposite directions, North Atlantic (RT3) and Euroasia (RT1). / digitalisering@umu.se

Tick-borne diseases represent major public and animal health issues worldwide. Ixodes ricinus, primarily associated with deciduous and mixed forests, is the principal vector of causative agents of viral, bacterial, and protozoan zoonotic diseases in Europe. Recently, abundant tick populations have been observed in European urban green areas, which are of public health relevance due to the exposure of humans and domesticated animals to potentially infected ticks. In urban habitats, small and medium-sized mammals, birds, companion animals (dogs and cats), and larger mammals (roe deer and wild boar) play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Presence of ticks infected with tick-borne encephalitis virus and high prevalence of ticks infected with Borrelia burgdorferi s.l., causing Lyme borreliosis, have been reported from urbanized areas in Europe. Emerging pathogens, including bacteria of the order Rickettsiales (Anaplasma phagocytophilum, “Candidatus Neoehrlichia mikurensis,” Rickettsia helvetica, and R. monacensis), Borrelia miyamotoi, and protozoans (Babesia divergens, B. venatorum, and B. microti) have also been detected in urban tick populations. Understanding the ecology of ticks and their associations with hosts in a European urbanized environment is crucial to quantify parameters necessary for risk pre-assessment and identification of public health strategies for control and prevention of tick-borne diseases.

Tick saliva contains a lot of biological active substances helping them to succesfully complete their feeding which is neccesary for their next development. Both proteinaceous and non-proteinaceous molecules including protease inhibitors are present in tick saliva. The biggest family of these proteases are serpins. Serpins are involved in many biological processes as blood coagulation, fibrinolysis, apoptosis or inflammation. The aim of this diploma work was to determine expression profiles of 10 serpins from nymphs of Ixodes ricinus fed for different times using quantitative real time PCR. For chosen genes (IRS 10, IRS 20) dsRNA for silencing of the gene was prepared and using RNA interference the role of these genes during tick (I. ricinus nymphs) feeding and transmission of Borrelia afzelii spirochetes, a vector of Lyme borreliosis, was evaluated.Tick saliva contains a lot of biological active substances helping them to succesfully complete their feeding which is neccesary for their next development. Both proteinaceous and non-proteinaceous molecules including protease inhibitors are present in tick saliva. The biggest family of these proteases are serpins. Serpins are involved in many biological processes as blood coagulation, fibrinolysis, apoptosis or inflammation. The aim of this diploma work was to determine expression profiles of 10 serpins from nymphs of Ixodes ricinus fed for different times using quantitative real time PCR. For chosen genes (IRS 10, IRS 20) dsRNA for silencing of the gene was prepared and using RNA interference the role of these genes during tick (I. ricinus nymphs) feeding and transmission of Borrelia afzelii spirochetes, a vector of Lyme borreliosis, was evaluated.