Infected Tick Salivary Gland Research Articles

A combined transcriptomic approach to identify candidates for an anti-tick vaccine blocking B. afzelii transmission

Ixodes ricinus is the vector for Borrelia afzelii, the predominant cause of Lyme borreliosis in Europe, whereas Ixodes scapularis is the vector for Borrelia burgdorferi in the USA. Transcription of several I. scapularis genes changes in the presence of B. burgdorferi and contributes to successful infection. To what extend B. afzelii influences gene expression in I. ricinus salivary glands is largely unknown. Therefore, we measured expression of uninfected vs. infected tick salivary gland genes during tick feeding using Massive Analysis of cDNA Ends (MACE) and RNAseq, quantifying 26.179 unique transcripts. While tick feeding was the main differentiator, B. afzelii infection significantly affected expression of hundreds of transcripts, including 465 transcripts after 24 h of tick feeding. Validation of the top-20 B. afzelii-upregulated transcripts at 24 h of tick feeding in ten biological genetic distinct replicates showed that expression varied extensively. Three transcripts could be validated, a basic tail protein, a lipocalin and an ixodegrin, and might be involved in B. afzelii transmission. However, vaccination with recombinant forms of these proteins only marginally altered B. afzelii infection in I. ricinus-challenged mice for one of the proteins. Collectively, our data show that identification of tick salivary genes upregulated in the presence of pathogens could serve to identify potential pathogen-blocking vaccine candidates.

Theileria sergenti and T. buffeli: Polymerase Chain Reaction-Based Marker System for Differentiating the Parasite Species from Infected Cattle Blood and Infected Tick Salivary Gland

Benign Theileria species in cattle, Theileria sergenti and T. buffeli, are morphologically indistinguishable. The polymerase chain reaction (PCR) was used to amplify the genes encoding the 33- and 34-kDa major piroplasm antigens (p33/34) of T. sergenti and T. buffeli from cattle blood infected with these parasites and tick salivary gland infected with T. sergenti. Following amplification, the p33 gene from T. sergenti and the p34 gene from T. buffeli were clearly differentiated using the restriction enzyme sites that were not shared between them. The oligonucleotide primer set, designed from the p33/34 genes, was specific for these Theileria species, since no amplification was detected with DNA from Babesia ovata, B. bovis, Anaplasma marginale, A. centrale, Eperythrozoon wenyoni, bovine white blood cells, and uninfected tick salivary glands. One tenth vol of the template prepared from either 25 μl of blood with 0.5% parasitemia or individual tick salivary glands with six infected acini allowed sufficient amplification for differentiation of the two parasite species by restriction enzyme digestion. In addition, this system could be used to demonstrate the simultaneous, experimentally induced infection of cattle with T. sergenti and T. buffeli. The PCR-based marker system therefore provides a means to differentiate T. sergenti from T. buffeli in infected cattle blood and infected tick salivary glands. This system may also be useful for the characterization of other benign Theileria species in cattle.

Conserved recombinant antigens of Anaplasma marginale and Babesia equi for serologic diagnosis

The competitive inhibition ELISA (CI-ELISA) format overcomes problems associated with antigen purity since the specificity of the CI-ELISA depends solely on the monoclonal antibody (mAb) used. Therefore, the CI-ELISA format is well suited for use with recombinant antigens. Molecular clones expressing a conserved 19 kDa protein of Anaplasma marginale and a 34 kDa protein of Babesia equi were derived and characterized. The 19 kDa A. marginale protein, conserved in all recognized Anaplasma species, and present in the infected tick salivary gland, was reactive with all bovine immune sera tested. The 34 kDa B. equi protein contains a protein epitope bound by antibody in equine immune sera from 19 countries. Monoclonal antibodies reactive with these proteins were derived and applied with recombinant copies of the 19 kDa A. marginale and 34 kDa B. equi proteins in a CI-ELISA format.

Identification of lambda gt11 clones encoding the major antigenic determinants expressed by Theileria parva sporozoites

An antiserum, C16, was raised in cattle against freeze-thawed extracts of sporozoites of Theileria parva (Muguga). This antiserum, which neutralizes sporozoite infectivity in vitro, identified theileria-specific antigens having approximate molecular masses of 105, 90, 85, 69, 67, 52, 47, and 43 kilodaltons (kDa) on Western blots (immunoblots) of infected tick salivary gland extracts. The antiserum was used to screen an expression library of T. parva (Muguga) genomic DNA fragments. Three recombinant bacteriophage clones carrying different theileria DNA inserts were studied. The expressed gene product from each clone was used to affinity purify antibodies from C16 antiserum for use in probing Western blots of uninfected and infected tick salivary gland extracts. The population of antibodies selected by each clone specifically recognized a subset of the antigens identified by C16 antiserum. The antigens fell into three distinct groups as defined by their reactivity with each set of selected antibodies. One group included antigens of 105, 90, 85, and 35 kDa, a second group included antigens of 69, 67, 52, 47, and 43 kDa, and the third group included an apparently distinct pair of antigens of 47 and 43 kDa. Thus, antibodies that reacted with determinants encoded by the three recombinant phage clones recognized all of the major antigens seen on Western blots probed with whole C16 antiserum. These results suggest that there may be only three immunodominant antigens expressed in T. parva (Muguga) sporozoites. Additionally, monoclonal antibodies have been raised which neutralize sporozoite infectivity in vitro. These antibodies react with epitopes of the antigens with Mrs of 69,000, 67,000, 52,000, 47,000, and 43,000 which are encoded in clone pgT-42 and have been used to localize these epitopes on the sporozoite surface.

Theileria parva: Expression of a sporozoite surface coat antigen

A monoclonal antibody specific for the Theileria parva sporozoite, which recognizes a determinant on the surface coat and blocks sporozoite infectivity, was used to investigate the presence of the determinant on other stages of the parasite lifecycle. Immunofluorescence techniques did not demonstrate this determinant on the kinete, schizont, merozoite, or piroplasm stages of the parasite. Immunoautoradiography, using a tritiated form of the monoclonal antibody, on sections of infected salivary glands collected from ticks that had fed for 0, 1, 2, 3, or 4 days revealed that the determinant recognized was synthesized predominantly during sporogony, between 2 to 3 days after the tick started feeding. Immunoelectron microscopy was performed on ultrathin frozen sections of infected tick salivary glands incubated with the monoclonal antibody followed by Protein-A--colloidal gold. The antigen or its precursor could be detected in the developing parasite. In ticks fed 2 days, the sporoblast was labeled, both in the cytoplasm and on parasite membranes, often including the nuclear envelope. In sections from ticks fed 4 days, the sporozoite surface membrane was labeled, as were membrane-bounded sporozoite organelles identified as micronemes. Observation by immunofluorescence, on sporozoites incubated with bovine peripheral blood lymphocytes, suggested that the antigen recognized by the monoclonal antibody does not enter the lymphocyte during sporozoite endocytosis. We conclude that synthesis of the antigen or its precursor(s) occurs during sporogony in the feeding tick, at the time of maximal parasite proliferation, and precedes the formation of morphologically mature sporozoites; the antigen's role in the parasite life cycle also appears to be limited to events associated with the sporozoite entry process.

A Rapid Method to Select Rhipicephalus appendiculatus Salivary Glands Infected with Theileria parva

Light microscopic studies on the detection and development of the salivary gland stages of Theileria parva in feeding adult ticks, Rhipicephalus appendiculatus, have involved the use of various stains such as Feulgen (Blewett and Branagan, 1973, Trop. Anim. Hlth Prod. 5:27-34), Giemsa (Pumell and Joyner, 1968, Parasitology 58: 725732) and Methyl Green Pyronin (MGP) (Walker et al., 1979, Trop. Anim. Hlth Prod. 11: 21-26; Irvin et al., 1981, Parasitology 82: 137-147). Although the method developed by Irvin et al. (1981, loc. cit.) significantly increased the speed with which parasites could be detected and examined, it still involved the dissection and teasing out of glands onto a microscope slide, followed by fixing, rinsing, staining, mounting in DPX under cover-slips and examination by a light microscope. We report here the development of a method which allows detection and selection of heavily infected salivary glands based on direct staining of a small part of 1 salivary gland in a drop of MGP. When adult ticks were immobilized in paraffin wax and the cuticle and gut removed, it was possible to fix and stain the salivary glands without removing them from the tick and thus detect T. parva-infected salivary gland acini using a dissection microscope at x 50 magnification. Carnoy's fixative (absolute ethanol 60%, chloroform 30%, glacial acetic acid 10%), was placed on each dissected tick for 5 min, followed by a rinse with 70% ethanol and one rinse with distilled water for 2 min each. Staining was carried out with a drop of MGP (aqueous Methyl Green 0.04%, aqueous Pyronin Y 0.02% in 0.5 M acetate buffer, pH 4.8) for 8 min. Nuclei of uninfected acinar cells stained blue whereas their cytoplasm containing RNA stained pink. The cytoplasm of infected cells containing parasite nuclear chromatin stained blue, allowing them to be distinguished from uninfected cells. In attempts to further simplify the method, it became clear that fixation prior to staining was not necessary for detection of the parasite. This observation then led to the development of a protocol for the selection of heavily infected salivary glands. Salivary glands infected with T. parva were obtained by feeding R. appendiculatus nymphs on cattle reacting to stabilate-induced infection (Cunningham et al., 1973, Int. J. Parasit. 3: 583587). Four-day fed adult female and male ticks were mounted in paraffin wax in a Petri dish and individual ticks numbered in the wax near the mouthparts. As many as 100 ticks could be mounted in 1 Petri dish. The dissection medium usually consisted of Dulbecco's phosphate buffered saline pH 7.4 (PBS), but other media such as Hanks' Balanced Salt Solution, Dulbecco's Minimum Essential Medium or Leibovitz's L1 5 could also be used. The dorsum and gut of the ticks were removed and the dissecting medium replaced by fresh PBS. A small piece of salivary gland was removed from every tick and deposited in a drop of PBS in a numbered well in the top row of a Wellcome PTFE Multispot slide (C. A. Hendley, Essex, UK), the numbers of the wells corresponding with the numbers of the ticks. The wells in the bottom row of the slide contained a drop of MGP. All salivary gland pieces were then transferred to the wells in the bottom row containing the MGP and left to stain for 8 min before transfer back to the original drops of PBS on the top row of the slide. Glands were examined and their infection level assessed. Salivary gland pieces from 15 to 20 ticks were routinely processed at a time. The numbers of the glands that contained many infected acini were recorded. When screening of ticks was completed and the numbers of all heavily infected ticks recorded, the remainder of the gland from which a sample was taken, together with the second gland, were removed for experimental use. Particular advantages of this method are its rapid process and minimal risk of losing or damaging the infected glands. This technique has been successfully applied for the selection of heavily infected tick salivary glands used for immunizing mice for the production of anti-sporozoite mon-