A reverse vaccinology approach to the identification and characterization of Ctenocephalides felis candidate protective antigens for the control of cat flea infestations.

BACKGROUND: Despite the abundance of the domestic cat flea, Ctenocephalides felis (Bouché, 1835) and disease risks associated with them, flea control is difficult and requires the development of new control interventions such as vaccines. In this study, a reverse vaccinology approach was designed to achieve a rational selection of cat flea candidate protective antigens. METHODS: Based on transcriptomics and proteomics data from unfed adult fleas it was possible to select more specific candidate protective antigens based on highly represented and functionally relevant proteins present in the predicted exoproteome. The protective capacity of the recombinant antigens was evaluated for the control of C. felis infestations in vaccinated cats. RESULTS: Vaccination with recombinant antigens induced an antibody response in immunized cats. Furthermore, a correlation was obtained between the effect of vaccination (antibody levels) and vaccine efficacy on flea phenotype (egg hatchability). The results suggested that the main effect of vaccination with these antigens was on reducing cat flea egg hatchability and fertility, with an overall vaccine efficacy of 32-46%. Although vaccination with these antigens did not have an effect on flea infestations, vaccines affecting reproductive capacity could reduce cat flea populations, particularly under conditions of direct insect transmission between cats. CONCLUSIONS: These results support the development of vaccines with protective antigens affecting flea reproduction and development after feeding on immunized animals for the control of cat flea infestations.

Identification of genes associated with blood feeding in the cat flea, Ctenocephalides felis.

BACKGROUND: The cat flea (Ctenocephalides felis) is a blood-feeding ectoparasitic insect and particular nuisance pest of companion animals worldwide. Identification of genes that are differentially expressed in response to feeding is important for understanding flea biology and discovering targets for their control. METHODS: C. felis fleas were maintained and fed for 24 h using an artificial rearing system. The technique of suppression subtractive hybridization was employed to screen for mRNAs specifically expressed in fed fleas. RESULTS: We characterized nine distinct full-length flea transcripts that exhibited modulated or de novo expression during feeding. Among the predicted protein sequences were two serine proteases, a serine protease inhibitor, two mucin-like molecules, a DNA topoisomerase, an enzyme associated with GPI-mediated cell membrane attachment of proteins and a component of the insect innate immune response. CONCLUSIONS: Our results provide a molecular insight into the physiology of flea feeding. The protein products of the genes identified may play important roles during flea feeding in terms of blood meal digestion, cellular growth/repair and protection from feeding-associated stresses.

Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: gene identification, cloning, expression, assay development, identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse.

Glutamate decarboxylase (l-glutamate 1-carboxylyase, E.C. 4.1.1.15, GAD) is the rate-limiting enzyme for the production of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrates and invertebrates. We report the identification, isolation and characterization of cDNAs encoding GAD from the parasitic arthropods Ctenocephalides felis (cat flea) and Rhipicephalus microplus (cattle tick). Expression of the parasite GAD genes and the corresponding Drosophila melanogaster (fruit fly) GAD1 as well as the mouse GAD(65) and GAD(67) genes in Escherichia coli as maltose binding protein fusions resulted in functional enzymes in quantities compatible with the needs of high throughput inhibitor screening (HTS). A novel continuous coupled spectrophotometric assay for GAD activity based on the detection cascade GABA transaminase/succinic semialdehyde dehydrogenase was developed, adapted to HTS, and a corresponding screen was performed with cat flea, cattle tick and fruit fly GAD. Counter-screening of the selected 38 hit substances on mouse GAD(65) and GAD(67) resulted in the identification of non-specific compounds as well as inhibitors with preferences for arthropod GAD, insect GAD, tick GAD and the two mouse GAD forms. Half of the identified hits most likely belong to known classes of GAD inhibitors, but several substances have not been described previously as GAD inhibitors and may represent lead optimization entry points for the design of arthropod-specific parasiticidal compounds.